Receptors providing targeted costimulation for adoptive cell therapy

ABSTRACT

The present invention relates to a chimeric costimulatory antigen receptor (CoStAR) useful in adoptive cell therapy (ACT), and cells comprising the CoStAR. The CoStAR can act as a modulator of cellular activity enhancing responses to defined antigens. The present invention also provides CoStAR proteins, nucleic acids encoding the CoStAR and therapeutic uses thereof.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is related to U.S. Patent Application Ser. No.63/053,494 filed Jul. 17, 2020, the contents of which are incorporatedherein by reference in their entireties.

Reference is made to GB patent application Serial No. 1900858.0, filed22 Jan. 2019, U.S. patent application Ser. No. 62/951,770, filed 20 Dec.2019, International application PCT/GB2020/050120, filed 20 Jan. 2020,and U.S. provisional patent application 63/053,498, filed Jul. 17, 2020.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSeqListing_INSTB.005C1.txt created on Jun. 30, 2021, which is 349,897bytes in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a chimeric costimulatory antigenreceptor (CoStAR) useful in adoptive cell therapy (ACT), and cellscomprising the CoStAR. The CoStAR can act as a modulator of cellularactivity enhancing responses to defined antigens. The present inventionalso provides CoStAR proteins, nucleic acids encoding the CoStAR andtherapeutic uses thereof.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using autologous T-cells to mediate cancerregression has shown much promise in early clinical trials. Severalgeneral approaches have been taken such as the use of naturallyoccurring tumor reactive or tumor infiltrating lymphocytes (TILs)expanded ex vivo. Additionally, T-cells may be genetically modified toretarget them towards defined tumor antigens. This can be done via thegene transfer of peptide (p)-major histocompatibility complex (MHC)specific T-cell Receptors (TCRs) or synthetic fusions between tumorspecific single chain antibody fragment (scFv) and T-cell signalingdomains (e.g. CD3ζ), the latter being termed chimeric antigen receptors(CARs).

TIL and TCR transfer has proven particularly good when targetingmelanoma (Rosenberg et al. 2011; Morgan 2006), whereas CAR therapy hasshown much promise in the treatment of certain B-cell malignancies(Grupp et al. 2013).

Costimulatory signals are useful to achieve robust CAR T cell expansion,function, persistence and antitumor activity. The success of CAR therapyin leukemia has been partly attributed to the incorporation ofcostimulatory domains (e.g. CD28 or CD137) into the CAR construct,signals from which synergize with the signal provided by CD3ζ to enhanceanti-tumor activity. The basis of this observation relates to theclassical signal 1/signal 2 paradigm of T-cell activation. Here signal1, provided by the TCR complex, synergizes with signal 2 provided bycostimulatory receptors such as CD28, CD137 or CD134 to permit the cellsto undergo clonal expansion, IL-2 production and long term survivalwithout the activation induced cell death (AICD) associated with signal1 alone. Furthermore the involvement of signal 2 enhances the signalgenerated through signal 1 allowing the cells to respond better to lowavidity interactions such as those encountered during anti-tumorresponses.

Targeted costimulation will have beneficial effects for non-CAR-basedT-cell therapies. For example, incorporating costimulatory domains intoa chimeric TCR has been shown to enhance responses of T-cells towardspMHC (Govers 2014). While tumor infiltrating lymphocytes (TILs) utilizetheir endogenous TCRs to mediate tumor recognition, it has not beenpossible to engineer the endogenous TCR. Thus TIL are subject tosubstantial limitations as tumor cells express very few costimulatoryligands. The ability to induce targeted costimulation of TIL, or indeedany other adoptive T-cell therapy product, would be beneficial.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention provides novel chimeric costimulatory antigen receptors(CoStARs) and cells comprising or expressing the CoStARs which arebeneficial for CAR and non-CAR based T-cell therapies alike. The presentinvention uses cells that express a novel chimeric costimulatoryreceptor to provides a costimulatory signal to T-cells upon engagementwith a defined disease-associated, for example tumor-associated,antigen.

There have been several reports in which split signal 1 and signal 2have been used to drive antigen specific responses in engineered T-cells(Alvarez-Vallina & Hawkins 1996). However, none have utilized the fulllength CD28 molecule. There are specific advantages to using full lengthreceptors, such as CD28 as opposed to truncated forms. A full lengthreceptors may be capable of dimerization, enabling the receptor tofunction in its native form, indeed chimeric antigen receptors fail tofunction optimally when expressed as a monomer (Bridgeman et al. 2010).

In an embodiment, a CoStAR of the invention induces signal 2 uponengagement with a defined antigen such as a disease associated or tumorassociated antigen. A full length CD28 molecule contains motifs criticalto its native function in binding members of the B7 family of receptors;although this is potentially dangerous from the perspective of CARscarrying CD28 and CD3ζ receptors in tandem, wherein ligation of CAR byB7 could trigger T-cell activation, there are beneficial qualities forreceptors harboring signal 2 receptors alone. In an aspect, theinvention provides a targeted chimeric costimulatory receptor (CoStAR)which comprises an extracellular antigen binding domain operativelylinked to a transmembrane domain, a first signaling domain, and a CD40signaling domain or a signaling fragment thereof. The inventors havediscovered that costimulatory receptors comprising a CD40 signalingdomain display novel and improved activity profiles.

In certain embodiments of the invention, the CD40 signaling domaincomprises SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In certainembodiments, the CD40 signaling fragment comprises, consists, orconsists essentially of an SH3 motif (KPTNKAPH (SEQ ID NO:26), PTNKAPHP(SEQ ID NO:118) or PTNKAPH(SEQ ID NO:119), TRAF2 motif (PKQE (SEQ IDNO:27), PKQET (SEQ ID NO:120), PVQE (SEQ ID NO:28), PVQET (SEQ IDNO:121), SVQE (SEQ ID NO:29), SVQET (SEQ ID NO:122)), TRAF6 motif(QEPQEINFP (SEQ ID NO:30) or QEPQEINFP (SEQ ID NO:123)), PKA motif(KKPTNKA (SEQ ID NO:31), SRISVQE (SEQ ID NO:32), or a combinationthereof, or is a full length CD40 intracellular domain. In certainembodiments, one or more of the SH3, TRAF2, TRAF6, or PKA motifs of theCD40 signaling domain is mutated. In certain embodiments, one or more ofthe SH3, TRAF2, TRAF6, or PKA motifs of the CD40 signaling domain ispresent in multiple copies.

In certain embodiments, the first signaling domain of the CoStARcomprises a signaling domain or signaling fragment of a receptor, suchas, for example a tumor necrosis factor receptor superfamily (TNFRSF)receptor, including but not limited to CD2, CD9, CD26, CD27, CD28, CD29,CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100,CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS),CD357 (GITR), or EphB6. In certain embodiments, the CoStAR comprisesCD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55,CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM),CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. In embodiments,wherein the first signaling domain comprises a CD40 signaling domainthus the CoStAR comprises elements of two CD40 signaling domains.

In certain embodiments, the CoStAR comprises a second signaling domainor signaling fragment of a receptor, such as, for example a tumornecrosis factor receptor superfamily (TNFRSF) receptor, including butnot limited to CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46,CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB),CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6. Thefirst signaling domain or signaling fragment, the CD40 signaling domainor signaling fragment, and the second signaling domain or signalingfragment can be in any order. Exemplary embodiments include, withoutlimitation, CoStAR which comprise CD28, CD137, and CD40 signalingdomains, CD28, CD134, and CD40 signaling domains, CD28, CD2, and CD40signaling domains, CD28, GITR, and CD40 signaling domains, CD28, CD29,and CD40 signaling domains, or CD28, CD150, and CD40 signaling domains.

In certain embodiments a CoStAR of the invention is engineered not toprovide signal 1. Accordingly, in certain embodiments, a CoStAR of theinvention does not comprise a signal 1 signaling domain. In certainembodiments, a CoStAR of the invention does not comprise a CD3ζsignaling domain.

In certain embodiments, a CoStAR of the invention is engineered toprovide signal 2 in a cell that is capable of providing signal 1 uponantigen binding (e.g., a T cell receptor provides signal 1 upon antigenengagement). In certain embodiments, a CoStAR of the invention isengineered to provide signal 2 in a cell in response to antigen-specificbinding by the CoStAR when the antigen is on the surface of a targetcell. In certain embodiments, a CoStAR of the invention is engineerednot to provide signal 2 in a cell in response to antigen-specificbinding by the CoStAR when the antigen is soluble and not attached tothe surface of a target cell.

In certain embodiments, the extracellular binding domain of a CoStAR ofthe invention is operatively linked to the transmembrane domain by alinker and/or a spacer. In certain embodiments, the linker comprisesfrom about 5 to about 20 amino acids. In certain embodiments, the linkercomprises AAAGSGGSG (SEQ ID NO:8).

In certain embodiments, a CoStAR of the invention comprises a spacerwhich operatively links the extracellular binding domain to thetransmembrane domain and comprises from about 10 to about 250 aminoacids. In certain embodiments, the spacer comprises an extracellularsequence of CD8 or CD28 or a fragment thereof. In certain embodiments,the CoStAR comprises a second extracellular binding domain. In certainembodiments, the second binding domain comprises an extracellular ligandbinding domain from CD8 or CD28. In certain embodiments, the spacercomprises one or more immunoglobulin domains or an immunoglobulinconstant region. In certain embodiments, the spacer comprises one ormore immunoglobulin domains or an immunoglobulin constant region of SEQID NO:13.

In certain embodiments the transmembrane domain of a CoStAR of theinvention comprises a transmembrane domain of a TNFRSF protein. Incertain embodiments, a transmembrane domain of a CoStAR of the inventioncomprises a transmembrane domain of CD28 or CD8. In certain embodiments,a transmembrane domain of a CoStAR of the invention comprises atransmembrane sequence of SEQ ID NO:11 or SEQ ID NO:12.

The CoStARs of the invention are useful to stimulate immune an immuneresponse against a selected target. In certain embodiments, a CoStAR ofthe invention comprises an extracellular binding domain that binds to atumor associated antigen. In certain embodiments, a CoStAR of theinvention comprises an extracellular binding domain that binds to atumor microenvironment associated antigen. In certain embodiments, theCoStAR comprises two or more extracellular binding domains. In certainembodiments, the extracellular binding domain binds to CD70, CD146,FOLR1, carcinoembryonic antigen (CEA), 5T4, mellanotransferrin (CD228),Her2, EGFR, GPC3, melanoma-associated chondroitin sulphate proteoglycan(MCSP/CSPG4), CD71, EPCAM, SM5-1, folate receptor or CA125, PDL-1, CD155PD-1, mesothelin, or a tumor specific peptide (p)-majorhistocompatibility (MHC) complex, or a tumor specific pMHC complexantigen specific single chain T-cell receptor (scTCR), or transferrin,or an antibody or antigen binding protein.

In certain embodiments wherein the binding domain binds to PDL1, theCoStAR comprises SEQ ID NO:6. In certain embodiments wherein theextracellular binding domain binds to CEA, the CoStAR comprises SEQ IDNO:5. In certain embodiments wherein the extracellular binding domainbinds to FOLR1, the CoStAR comprises SEQ ID NO:4. In certain embodimentswherein the binding domain binds to CD155, CD112 or CD113, the CoStARcomprises SEQ ID NO:7.

According to the invention, an extracellular binding domain cancomprise, without limit, an scFv, a peptide, an antigen binding portionof an antibody, an antibody heavy-chain, a ligand of a target receptoror a ligand binding portion of a receptor.

In certain embodiments, a CoStAR of the invention comprises a CD3ζsignaling domain, for example located at the C-terminus.

In certain embodiments, a CoStAR of the invention comprises anN-terminal signal peptide.

In an aspect of the invention, there is provided a nucleic acid whichencodes a CoStAR of the invention. The nucleic acid may be optimized,for example be codon optimized for expression in a host cell. In anon-limiting embodiment, the nucleic acid is codon optimized forexpression in a human cell.

In an aspect of the invention, there is provided vector which encodesand is capable of expressing a CoStAR of the invention.

In an aspect of the invention, there is provided a cell which expressesa CoStAR of the invention. In certain embodiments, the cell expresses aCoStAR that binds to FOLR1. In other embodiments, the cell expresses aCoStAR that binds to CA125.

In an aspect, the invention provides a cell which expresses a CoStARthat is specific for FOLR1 wherein the cell is activated when the CoStARreacts with or binds to FOLR1 on the surface of a target cell but notwhen the CoStAR binds to or reacts with soluble FOLR1. In an embodiment,the cell is a T cell or a TIL that expresses a T cell receptor or otherreceptor specific for a tumor associated antigen expressed by the targetcell.

In other embodiments, the cell expresses a CoStAR specific for PDL1. Inother embodiments, the cell expresses a CoStAR specific for CEA. Incertain embodiments, the cell expresses two or more CoStARs of theinvention. In a particular embodiment, the cell expresses a CoStAR thatbinds to FOLR1 and a CoStAR that binds to CA125, such as but not limitedto anti-FOLR1.CD28.CD40 and anti-CA125.41BB.CD40. In a particularembodiment, the cell expresses a CoStAR which binds to FOLR1 and aCoStAR which binds to PDL1, such as but not limited toanti-FOLR1.CD28.CD40 and PD1.CD28.CD40.

In certain embodiments, a cell engineered to express a CoStAR of theinvention comprises an alpha-beta T cell, gamma-delta T cell, Tregulatory cell, TIL, NKT cell or NK cell. In certain embodiments, acell engineered to express a CoStAR of the invention coexpresses achimeric antigen receptor (CAR) or a T cell receptor (TCR).

In an aspect, the invention provides a method of making the cell whichexpresses a CoStAR which comprises transducing or transfecting a cellwith a vector which encodes and is capable of expressing a CoStAR of theinvention.

The invention provides a method for preparing a population of cells thatexpress a CoStAR of the invention by transducing or transfecting cells,detecting expression of the CoStAR and enriching, expanding, and/orselecting cells that express the CoStAR.

In an aspect, the invention provides a method of treating a disease in asubject by administering a population of cells which express a CoStAR ofthe invention.

In an aspect, the invention provides a method of preparing TILcomprising disaggregating a resected tumor to obtain a refined resectedtumor product, performing a first expansion by culturing the refinedresected tumor product in a cell culture medium comprising IL-2 toproduce a first population of TILs, performing a second expansion byculturing the first population of TILs with additional IL-2, OKT-3, andantigen presenting cells (APCs), to produce a second population of TILs;and harvesting and/or cryopreserving the second population of TILs,wherein the method comprises transfecting or transducing the TILs toexpress a CoStAR of the invention. In an embodiment, the tumor comprisesan ovarian tumor. In an embodiment, the tumor comprises a renal tumor.In an embodiment, the tumor comprises a lung tumor.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1—Structural organisation of single costimulatory and fusioncostimulatory domain receptors. A schematic representation of CoStARreceptors set out in the claims is shown. First a CoStAR based on asingle costimulatory receptor, and secondly a fusion CoStAR consistingof a full length costimulatory receptor signalling domain fused to asecond costimulatory domain.

FIGS. 2A-2G—Genomic organisation of potential CoStAR configurations—TheCoStAR consists of an antigen binding domain, an optional spacer domainand a costimulatory domain as shown in figure and described in claims.The CoStAR may be expressed as shown; alone from a promoter with theCoStAR consisting of a single (FIG. 2A) or fusion (FIG. 2B)costimulatory receptor; (FIG. 2C) may be expressed with an epitope tag(e.g. His tag, DYKDDDDK (SEQ ID NO:14) etc) at the N or C-terminus toenable direct staining of the CoStAR; (FIG. 2D) along with a marker geneseparated using a 2A cleavage sequence or internal ribosomal entry site(IRES); (FIG. 2E) along with a marker gene which is expressed from asecond promoter; (FIG. 2F) along with a protein of interest such as achimeric antigen receptor or T-cell receptor separated using a 2Acleavage sequence or internal ribosomal entry site (IRES); (FIG. 2G)along with a protein of interest such as a chimeric antigen receptor orT-cell receptor which is expressed from a second promoter. It would beclear to an individual with sufficient knowledge that the CoStAR andmarker gene/chimeric antigen receptor/T-cell receptor/other protein ofinterest could be expressed in either orientation or 3′ (3-prime) or 5′(5-prime) to one another.

FIGS. 3A-3E—Functional activity of CoStAR in T-cells in response toLS174T and LoVo tumor presented antigen. Normal donor T-cell populationsfrom donor 1 (FIGS. 3A & 3D), donor 2 (FIG. 3B) and donor 3 (FIGS. 3C &3E) were lentivirally engineered to express a CoStAR which targetscarcinoembryonic antigen and magnetically sorted to enrich for thetransgene using CD34 magnetic selection. T-cells were mixed withwild-type un-engineered CEA+ tumor cells (Non-activating tumor) or CEA+tumor cells engineered to express a cell surface anchored anti-CD3single chain antibody fragment (Activating tumor) at the indicatedeffector to target ratios and IL-2 measured in the supernatant by ELISA.Data obtained using LS174T cells (A, B & C) and LoVo (D & E).

FIGS. 4A-4D—Effect of CoStAR on T-cell proliferation. 5×10⁵ transducedand non transduced T-cells were mixed with 6.25×10³ wild-type LoVo orLoVo-OKT3 cells in the presence (FIG. 4A) or absence (FIG. 4B) of IL-2and cell counts made after three days. In another assay under the samecell ratios T-cells from two donors (FIGS. 4C and 4D) were loaded withproliferation dye and the number of proliferation cycles the cells hadgone through determined by dye dilution after six days using flowcytometry.

FIG. 5—IL-2 activity of CoStAR fusion receptors in primary humanT-cells. Normal donor CD8+ T-cells from seven donors (except controlCoStAR is three donors) were lentivirally transduced with the indicatedCEA-targeting CoStARs and IL-2 production assessed after an overnightstimulation in the presence of LoVo-OKT3 cells. The proportion of IL-2positive cells was determined using intracellular flow staining in boththe CD34 negative (CoStAR non-transduced) and CD34+ (CoStAR transduced)populations. Asterisks show significant differences between thetransduced and non-transduced populations using paired Wilcoxon signedrank test with * p<0.05

FIGS. 6A-6D—Multi parameter analysis of CoStAR activity in primary humanT-cells. Normal donor CD8+ T-cells were lentivirally transduced with theindicated CEA-targeting CoStARs and IL-2 production assessed after anovernight stimulation in the presence of LoVo-OKT3 cells. The proportionof IL-2 (seven donors) (FIG. 6A), IFNγ (seven donors) (FIG. 6B), bcl-xL(five donors) (FIG. 6C) and CD107a (six donors) (FIG. 6D) positive cellswas determined using intracellular flow staining in both the CD34negative (CoStAR non-transduced) and CD34+ (CoStAR transduced)populations. Control is an irrelevant CA125 targeting CoStAR and is fromthree donors in all instances. Heat maps are averages of all donors withthe intensity of colour related to the percentage of cells positive fora particular read out under the defined conditions.

FIG. 7—CD40 enhances IL-2 production from CD28-based CoStARs. Primaryhuman T-cells from three healthy donors were left non-transduced ortransduced with either extracellular domain truncated CD28 (Tr CD28),full length CD28 (FL CD28), or CD28.CD40-based CoStARs harboring a CEAspecific scFv (MFE23). Transduced cells were selected using a CD34marker gene and expanded prior to analysis. T-cells were mixed at an 8:1effector to target ratio with OKT3 expressing CEA+ LoVo cells for 20hours before analysis of IL-2 production by ELISA.

FIG. 8—Effect of signalling domain and target antigen on CoStAR-mediatedT-cell expansion. T-cells were transduced with either DYKDDDDK (SEQ IDNO:14) epitope-tagged CD28 or CD28.CD40 based CoStARs harboring CA125,FOLR1 or CEA specific scFv, or FOLR1 specific binding peptide (C7).T-cells were mixed with OKT3 expressing, CA125+/FOLR1+/CEA− cell lineOVCAR3. The number of transduced cells were counted every 7 days up to21 days, with fresh OVCAR3 cells added following each count.

FIG. 9—CD40 based CoStARs enhance costimulation of T-cells in a model ofTCR-transfer. Primary human T-cells from three healthy donors weretransduced with a CEA specific TCR plus either a DYKDDDK-tagged CD28 orCD28.CD40 based CoStAR harboring either an MFE23 (open or closedcircles) or CA125 (open squares) specific scFv. T-cells were mixed at a1:1 effector:target ratio with CEA+/CA125− H508 cells and intracellularcytokine staining performed to determine the number of responding CD4+or CD8+ T-cells in the TCR+/CoStAR+, TCR+/CoStAR−, TCR−/CoStAR+ andTCR−/CoStAR− populations. A 2-way ANOVA (Tukeys test) was performed todetermine significant differences in activity: *p>0.05, ** p>0.01, ***p>0.001, **** p>0.0001.

FIGS. 10A-10B—CoStAR dependent enhancement of activity in a model of TCRtransfer. Primary human T-cells from three healthy donors weretransduced with a CEA specific TCR plus either a DYKDDDK-tagged CD28 orCD28.CD40 based CoStAR harboring either an MFE23 (open or closedcircles) or CA125 (open squares) specific scFv. T-cells were mixed at a1:1 effector:target ratio with CEA+/CA125− H508 cells and intracellularcytokine staining performed to determine the number of respondingCD4+(FIG. 10A) or CD8+(FIG. 10B) T-cells in the TCR+/CoStAR+,populations. A 2-way ANOVA (Tukeys test) was performed to determinesignificant differences in activity: ** p>0.01, **** p>0.0001.

FIG. 11 depicts enrichment and expansion of primary human T-cellstransduced to express costimulatory molecules of the invention. MFE23 isa single chain Fv antibody that has a high affinity for carcinoembryonicantigen (CEA). Primary human T-cells were mock transduced or transducedwith MFE23.CD28 or MFE23.CD28.CD40 CoStAR, each harboring a CD34 markergene separated by a 2A cleavage peptide. Following in vitro culturecells were enriched for CD34 using MACS™ paramagnetic selection reagents(Miltenyi Biotech) and then the cells expanded in number usingirradiated feeder cells. Exemplary plots from one of three donors isshown.

FIGS. 12A-12D depict expansion of T-cells transduced with costimulatorymolecules of the invention in response to stimulation and exogenousIL-2. Cells were mock transduced or transduced with MFE23.CD28 orMFE23.CD28.CD40 CoStAR and cocultured with LoVo-OKT3 cells at an 8:1effector:target ratio in the presence (200 IU/ml) or absence ofexogenous IL-2. At days 1, 4, 7, 11 and 18 cells were taken and thenumber of viable T-cells enumerated by using anti-CD2 reagents on aMACSQuant flow cytometer. (FIG. 12A) In the absence of stimulation bytumor and IL-2 cells declined in number as would be expected. (FIG. 12B)In the absence of stimulation but presence of IL-2 there was a moreapparent survival of the cells, but no specific growth. (FIG. 12C) Inthe presence of tumor, but absence of IL-2 mock cells did not showspecific survival. MFE23.CD28 CoStAR mediated an apparent doubling inexpansion over the first four days followed by decline. MFE23.CD28.CD40mediated a greater expansion up to day 7 followed by a steady decline.(FIG. 12D) Under the same conditions but in the presence of IL-2 bothmock and MFE23.CD28 transduced cells demonstrated a 20-fold expansionover 18 days, whereas MFE23.CD28.CD40 cells expanded by over 60-fold.Thus CD28.CD40 based receptors demonstrate superior expansion andsurvival under conditions of stimulation both in the presence andabsence of exogenous IL-2.

FIGS. 13A-13M depict cytokine production by mock, MFE23.CD28 orMFE23.CD28.CD40 engineered T-cells. Bead array analysis was performed onsupernatants obtained from T-cell/tumor cocultures. Engineered T-cellswere incubated at a 1:1 effector:target ratio with LoVo-OKT3 cells for24 hours and supernatant collected. Conditioned supernatant was alsocollected from an equal number of T-cells alone, or LoVo-OKT3 cellsalone. Cytokine production was analysed using a Legendplex™ HumanTH1/TH2 cytokine panel (Biolegend). (FIG. 13A) IL-2; (FIG. 13B) IFN-γ;(FIG. 13C) TNFα; (FIG. 13D) IL-4; (FIG. 13E) IL-5; (FIG. 13F) IL-13;(FIG. 13G) IL-17A; (FIG. 13H) IL-17F; (FIG. 13I) IL-22; (FIG. 13J) IL-6;(FIG. 13K) IL-10; (FIG. 13L) IL-9; (FIG. 13M) IL-21. Cytokines wereeither very low or undetectable in media from T-cells or tumor alone.When cocultured with tumor, cytokine production was enhanced. MFE23.CD28enhanced production of IL-2, IL-5, IL-17A/17F, IL-10, IL-9 and IL-21compared to mock. MFE23.CD28.CD40 also enhanced production of TNFα,IL-13 and IL-22. MFE23.CD28.CD40 and further enhanced the production ofa number of cytokines greater than that provided by MFE23.CD28 (IL-2,IL-9 and IL-17F), as well as reducing the production of some cytokinesbelow the levels seen with MFE23.CD28 (IL-5 and IL-10). Together thisdata demonstrates that addition of CD40 to CD28-based costimulatoryreceptors enhances and/or modulates their specific activity with respectto cytokine production.

FIGS. 14A-14M depict an analysis of chemokines using a Legendplex™ HumanPro inflammatory chemokine panel. (FIG. 14A) IL-8 (CXCL8); (FIG. 14B)IP-10 (CSCL10); (FIG. 14C) Eotaxin (CCL11); (FIG. 14D) TARC (CCL17);(FIG. 14E) MCP-1 (CCL2); (FIG. 14F) RANTES (CCL5); (FIG. 14G) MIP-1a(CCL3) (FIG. 14H) MIG (CXCL9) (FIG. 14I) ENA-78 (CXCL5) (FIG. 14J)MIP-3a (CCL20) (FIG. 14K) GROα (CXCL1) (FIG. 14L) I-TAC (CXCL11) (FIG.14M) MEP-1β (CCL4). Chemokines were either very low or undetectable inmedia from T-cells alone. When cocultured with tumor, chemokineproduction was enhanced. MFE23.CD28 enhanced production of CXCL5,CXCL10, CXCL11, CCL17 and CCL20 compared to mock. MFE23.CD28.CD40 alsoenhanced production of CCL2, CXCL1 and CXCL9. MFE23.CD28.CD40 furtherenhanced the production of a number of cytokines greater than thatprovided by MFE23.CD28 (CXCL1, CXCL9, CXCL10, CXCL11, CCL17, CCL2,CXCL9, CCL5 and CCL20), as well as reducing the production of somecytokines below the levels seen with MFE23.CD28 (CCL4). Together thisdata demonstrates that addition of CD40 to CD28-based costimulatoryreceptors enhances and/or modulates their specific activity with respectto chemokine production.

FIGS. 15A-15H depict functional activity of ovarian CoStAR engineeredcells using a CoStAR harboring a FolR or CA125 reactive scFv (MOV19 &196-14 respectively). Human folate receptor alpha (FolR) represents asuitable target for a number of tumors including ovarian, head and neck,renal and lung and CA125 represents an alternative target for ovariancancer. Primary human T-cells from six healthy donors were engineeredwith either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 or MOV19.CD28.CD40receptors, all harboring a DYKDDDDK epitope tag for detection.Transduced cells were mixed with FolR+/CA125+ OvCAR-OKT3 cells beforeanalysis of effector activity using intracellular staining in theepitope tag positive and negative populations. Specific enhancement ofeffector activity determined by production of IL-2 (15A and 15B), TNFα(15C and 15D), CD137 (15E and 15F), and BCL-xL (15G and 15H) wasobserved in CD28 and CD28.CD40 engineered cells in response to bothCA125 and FolR, except for specific BCL-xL induction by MOV19.CD28 whichwas not observed compared to MOV19.CD28.CD40.

FIGS. 16A-16F depict three TIL populations mock transduced or engineeredwith MOV19.CD28.CD40 CoStAR and then mixed with patient matched tumordigest. The donor tumors displayed varying levels of FolR on the digest,ranging from negative (FIG. 16A), low expression (FIG. 16B) to highexpression (FIG. 16C). Mock and CoStAR negative TIL in the CoStARengineered populations of TIL matched for the FolR negative digestdemonstrated similar levels of CD137 upregulation following tumorcoculture which was not enhanced by the presence of CoStAR (FIG. 16D).In the TIL exposed to FolR low expressing digest there was anenhancement in activity in the CoStAR+ cells compared to CoStAR−, withCD137 expression increasing from <10% to >20% (FIG. 16E). In the TILexposed to FolR high tumor digest there was an increase in activity fromaround 20% in the CoStAR− population, up to approximately 50% in theCoStAR+ population (FIG. 16F).

FIGS. 17A-17C depict enhancement of effector functions. A FolR targetingCoStAR enhanced CD137 expression from ˜20% to ˜50% (FIG. 17A), TNFαproduction from 10% to 15% (FIG. 17B) and IL-2 production from 2% to 5%.(FIG. 17C) in response to FolR+ tumor digest.

FIGS. 18A-18F depict soluble ligand does not inhibit effector functions.T-cells from three healthy donors were engineered with MOV19.CD28 orMOV19.CD28.CD40 CoStAR and activated with either immobilised OKT3,providing stimulation in the absence of FolR, or with OvCAR-OKT3, toprovide TCR and CoStAR activity. Bcl-XL activity was increased frombetween 10 and 20% across the three donors following OKT3 stimulation(FIG. 18A) whereas IL-2 was increased between 0 and 12% (FIG. 18B) andTNFα increased between 0 and 20% (FIG. 18C). The presence of exogenoussoluble FolR did not enhance any of these particular effector functions.In the presence of OvCAR-OKT3, Bcl-XL induction was enhanced by ˜20% inCD28 CoStAR but by ˜35% in CD28.CD40 CoStAR (FIG. 18D), IL-2 inductionwas enhanced by ˜20% in CD28 CoStAR but 30-50% in CD28.CD40 CoStAR (FIG.18E) and TNFα production was enhanced by 20-30% in CD28 CoStAR and25-50% in CD28.CD40 CoStAR (FIG. 18F). Exogenous soluble FolR did nothave an inhibitory effect on any of these effector functions.

FIG. 19 depicts exemplary CoStAR constructs. MFE23: scFv specific forcarcinoembryonic antigen (CEA). Costimulatory domains are identified.CTP188: SEQ ID NO:89; CTP189: SEQ ID NO:109; CTP190: SEQ ID NO:41;CTP191: SEQ ID NO:45; CTP192: SEQ ID NO:43; CTP193: SEQ ID NO:42;CTP194: SEQ ID NO: 33; CTP195: SEQ ID NO:110; CTP196: SEQ ID NO:111;CTP197 SEQ ID NO:112; CTP198: SEQ ID NO:113; CTP199: SEQ ID NO:114;CTP200: SEQ ID NO:115; CTP201: SEQ ID NO:116; CTP202: SEQ ID NO:117;CTP203: SEQ ID NO:49; CTP204: SEQ ID NO:59.

FIG. 20 depicts CD4+ and CD8+ subpopulations of CD40 CoStAR modified Tcells. T cells of four healthy donors were activated and transduced withvarious CD40 CoStARs with a CD34 marker or mock transduced. Cells wereenriched for their CD34 expression and expanded following the rapidexpansion protocol (REP). CD4 (light grey) and CD8 (grey) T cellphenotypes were assessed 10-11 days after REP using anti-humanCD4-PerCP-eF710, anti-human CD8-PE-Cy7, and anti-human CD3-FITC. Datashown as mean+/−SD, n=4 healthy donors

FIGS. 21A-21C depict increased amount of IL-2 in PD-1 fusion CD40 CoStARcompared to mock transduced T cells. Donor cells activated withDynabeads and transduced with CTP188, CTP189, CTP194 (FIG. 21A) ormock-transduced were enriched for CD34 (transduction marker) expression(FIG. 21B), expanded following the rapid expansion protocol (REP) andfrozen for subsequent experiments. After thaw, cells were rested for 3-4days in complete RPMI supplemented with IL-2. The viability and absolutecount were assessed after overnight IL-2 starvation using DRAQ-7 (1:200)by flow cytometry (Novocyte) and data were analysed using theNovoExpress 1.5.0 software. Transduced T cells were cocultured inabsence of IL-2 with LoVo (CCL-229™ from ATCC) or LoVo.OKT3.GFP tumorcells at 8:1 effector to target ratio. After 24 hours, supernatants werecollected and frozen. LoVo and LoVo.OKT3.GFP naturally express CEA andPD-L1 on their surface, conferring signal 2 through the CoStAR alone(LoVo) or associated with signal 1 (LoVo.OKT3.GFP) to the transduced Tcells. Cocultures were performed in triplicates and correspondingnegative (T cells alone, tumor cells alone) and positive (PMA+ionomycin)controls were included in the experiment. After thaw, secreted IL-2 andIFN-γ were detected by ELISA and the absorbance was measured using theFLUOstar Omega microplate reader and subsequently analysed with theOmega MARS 3.42 R5 software. Each dot represents the mean of triplicatesfor one donor. Note that negative controls (T cells alone, tumor cellsalone) were all below the detection range (#) (FIG. 21C).

FIG. 22 depicts PD-1 extracellular domain conferring a slightproliferation advantage to CD40 CoStAR transduced T cells whencocultured with LoVo.OKT3. Healthy donor T cells activated withDynabeads and transduced with CTP188, CTP189, CTP194 or mock-transducedwere enriched for CD34 expression, expanded following the rapidexpansion protocol (REP) and frozen for subsequent experiments. Afterthaw, cells were rested for 3-4 days in complete RPMI supplemented withIL-2 and their transduction rate was determined looking at the CD34marker gene expression. The viability and absolute count were assessedafter overnight IL-2 starvation using DRAQ-7 (1:200) by flow cytometry(Novocyte) and data were analysed using the NovoExpress 1.5.0 software.Transduced T cells were cocultured in absence of IL-2 for 6-8 days withLoVo.OKT3.GFP tumor cells at 8:1 effector to target ratio, changing halfof the culture medium every 3-4 days. LoVo.OKT3.GFP naturally expressesCEA and PD-L1 on their surface, conferring both signal 2 and signal 1(OKT3) to the transduced T cells. After 6-8 days, the viability andabsolute count were assessed, and live T cells were rechallenged for anadditional week with fresh LoVo.OKT3.GFP tumor cells as described above.At the end of the long-term coculture, the viability and absolute countwere measured, and the fold expansion was calculated. Data shown asmean+/−SEM of n≤3 donors analysed in triplicates.

FIGS. 23A-23C depict exhaustion profiles of PD-1 fusion CD40 CoStARtransduced T cells after tumor challenge. Healthy donor T cells wereactivated with Dynabeads and transduced with CTP188, CTP189, CTP194 ormock transduced. Cells were enriched for CD34 marker expression,expanded following the rapid expansion protocol (REP) and frozen forsubsequent experiments. After thaw, cells were rested for 3-4 days incomplete RPMI supplemented with IL-2. The viability and absolute countwere assessed after overnight IL-2 starvation using DRAQ-7 (1:200) byflow cytometry (Novocyte) and data were analysed using the NovoExpress1.5.0 software. Transduced T cells were cocultured in absence of IL-2for 6-8 days with LoVo.OKT3.GFP tumor cells at 8:1 effector to targetratio, changing half of the culture medium every 3-4 days. LoVo.OKT3.GFPnaturally expresses CEA and PD-L1 on their surface, conferring bothsignal 2 and signal 1 (OKT3) to the transduced T cells. After 6-8 days,the viability and the absolute count were assessed, and live T cellswere rechallenged for an additional week with fresh LoVo.OKT3.GFP tumorcells as described above. Exhaustion profiles (LAG-3 (FIG. 23A), PD-1(FIG. 23B), TIM-3 (FIG. 23C)) of transduced (CD34+(grey)) ornon-transduced (CD34− (white)) CD4 (upper panels) and CD8 (lower panels)T cells were assessed by flow cytometry and shown as mean+/−SD of n≤3donors.

FIGS. 24A-24B depict T cells transduced with CD28, CD137 and CD40 aloneCoStARs secrete higher amount of IL-2 following activation compared tomock transduced T cells. (FIG. 24A) Healthy donor T cells were activatedwith Dynabeads and transduced with CTP190, CTP191, CTP192, CTP193,CTP194 or mock transduced. The correlation between the expression ofCD34 marker gene and MFE23 scFv on the surface of transduced T cellsfrom one subject (lower left panel), was assessed 8 days followingtransduction by flow cytometry. Cells were then enriched for CD34 markerexpression, expanded following the rapid expansion protocol (REP) andfrozen for subsequent experiments. After thaw, cells were rested for 3-4days in complete RPMI supplemented with IL-2 and their transduction ratewas determined looking at the CD34 marker gene expression (lower rightpanel). (FIG. 24B) The viability and absolute count were assessed afterovernight IL-2 starvation using DRAQ-7 (1:200) by flow cytometry(Novocyte) and data were analysed using the NovoExpress 1.5.0 software.Transduced T cells were cocultured in absence of IL-2 with LoVo (CCL229™from ATCC) or LoVo.OKT3.GFP tumor cells at 8:1 effector to target ratio.After 24 hours, supernatants were collected and frozen. LoVo andLoVo.OKT3.GFP naturally express CEA and PD-L1 on their surface,conferring signal 2 through the CoStAR alone (LoVo) or associated withsignal 1 (LoVo.OKT3.GFP) to the transduced T cells. Cocultures wereperformed in triplicates and corresponding negative (T cells alone,tumor cells alone) and positive (PMA+ionomycin) controls were includedin the experiment. After thaw, secreted IL-2 and IFN-γ were detected byELISA and the absorbance was measured using the FLUOstar Omegamicroplate reader and subsequently analysed with the Omega MARS 3.42 R5software. Each dot represents the mean of triplicates for one donor.Note that negative controls (T cells alone, tumor cells alone) were allbelow the detection range (#)(FIG. 24B).

FIG. 25 depicts CD28 and CD137 endodomains conferring a proliferationadvantage to CD40 CoStAR transduced T cells when cocultured withLoVo.OKT3. Healthy donor T cells were activated with Dynabeads andtransduced with CTP190, CTP191, CTP192, CTP193, CTP194 or mocktransduced. Cells were enriched for CD34 marker expression, expandedfollowing the rapid expansion protocol (REP) and frozen for subsequentexperiments. After thaw, cells were rested for 3-4 days in complete RPMIsupplemented with IL-2. The viability and absolute count were assessedafter overnight IL-2 starvation using DRAQ-7 (1:200) by flow cytometry(Novocyte) and data were analysed using the NovoExpress 1.5.0 software.Transduced T cells were cocultured in absence of IL-2 for 6-8 days withLoVo.OKT3.GFP tumor cells at 8:1 effector to target ratio, changing halfof the culture medium every 3-4 days. LoVo.OKT3.GFP naturally expressesCEA and PD-L1 on their surface, conferring both signal 2 and signal 1(OKT3) to the transduced T cells. After 6-8 days, the viability andabsolute count were assessed, and live T cells were rechallenged for anadditional week with fresh LoVo.OKT3.GFP tumor cells as described above.At the end of the long-term coculture, the viability and absolute countwere measured, and the fold expansion was calculated. Data shown asmean+/−SEM of n≤3 donors analysed in triplicates.

FIGS. 26A-26C depict exhaustion profiles of transduced T cells withCD28, CD2, CD137 and CD40 alone CoStARs after tumor challenge. Healthydonor T cells were activated with Dynabeads and transduced with CTP190,CTP191, CTP192, CTP193, CTP194 or mock transduced. Cells were enrichedfor CD34 marker expression, expanded following the rapid expansionprotocol (REP) and frozen for subsequent experiments. After thaw, cellswere rested for 3-4 days in complete RPMI supplemented with IL-2. Theviability and absolute count were assessed after overnight IL-2starvation using DRAQ-7 (1:200) by flow cytometry (Novocyte) and datawere analysed using the NovoExpress 1.5.0 software. Transduced T cellswere cocultured in absence of IL-2 for 6-8 days with LoVo.OKT3.GFP tumorcells at 8:1 effector to target ratio, changing half of the culturemedium every 3-4 days. LoVo.OKT3.GFP naturally expresses CEA and PD-L1on their surface, conferring both signal 2 and signal 1 (OKT3) to thetransduced T cells. After 6-8 days, the viability and the absolute countwere assessed, and live T cells were rechallenged for an additional weekwith fresh LoVo.OKT3.GFP tumor cells as described above. Exhaustionprofiles (LAG-3 (FIG. 26A), PD-1 (FIG. 26B), TIM-3 (FIG. 26C)) oftransduced (CD34+ (grey)) or non-transduced (CD34− (white)) CD4 (upperpanels) and CD8 (lower panels) T cells were assessed by flow cytometryand shown as mean+/−SD of n≤3 donors.

FIGS. 27A-27B depict CD40 CoStAR TRAF-binding site mutations have adirect impact on the secretion of IL-2 and IFN-γ following activation.(FIG. 27A) Cells of three donors were activated with Dynabeads andtransduced with CTP194, CTP195, CTP196, CTP197, CTP198, CTP199, CTP200or mock transduced. Cells were enriched for CD34 marker expression,expanded following the rapid expansion protocol (REP) and frozen forsubsequent experiments. After thaw, cells were rested for 3-4 days incomplete RPMI supplemented with IL-2 and their transduction rate wasdetermined looking at the CD34 marker gene expression (A, lower panel).(FIG. 27B) The viability and absolute count were assessed afterovernight IL-2 starvation using DRAQ-7 (1:200) by flow cytometry(Novocyte) and data were analysed using the NovoExpress 1.5.0 software.Transduced T cells were cocultured in absence of IL-2 with LoVo(CCL-229™ from ATCC) or LoVo.OKT3.GFP tumor cells at 8:1 effector totarget ratio. After 24 hours, supernatants were collected and frozen.LoVo and LoVo.OKT3.GFP naturally express CEA and PD-L1 on their surface,conferring signal 2 through the CoStAR alone (LoVo) or associated withsignal 1 (LoVo.OKT3.GFP) to the transduced T cells. Cocultures wereperformed in triplicates and corresponding negative (T cells alone,tumor cells alone) and positive (PMA+ionomycin) controls were includedin the experiment. After thaw, secreted IL-2 and IFN-γ were detected byELISA and the absorbance was measured using the FLUOstar Omegamicroplate reader and subsequently analysed with the Omega MARS 3.42 R5software. Each dot represents the mean of triplicates for one donor.Note that negative controls (T cells alone, tumor cells alone) were allbelow the detection range (#).

FIG. 28 depicts the critical role of the PVQET TRAF-binding motif inlong term survival and proliferation of CD28.CD40 CoStAR transduced Tcells when cocultured with LoVo.OKT3. Cells of three donors wereactivated with Dynabeads and transduced with CTP194, CTP195, CTP196,CTP197, CTP198, CTP199, CTP200 or mock transduced. Cells were enrichedfor CD34 marker expression, expanded following the rapid expansionprotocol (REP) and frozen for subsequent experiments. After thaw, cellswere rested for 3-4 days in complete RPMI supplemented with IL-2. Theviability and absolute count were assessed after overnight IL-2starvation using DRAQ-7 (1:200) by flow cytometry (Novocyte) and datawere analysed using the NovoExpress 1.5.0 software. Transduced T cellswere cocultured in absence of IL-2 for 6-8 days with LoVo.OKT3.GFP tumorcells at 8:1 effector to target ratio, changing half of the culturemedium every 3-4 days. LoVo.OKT3.GFP naturally expresses CEA and PD-L1on their surface, conferring both signal 2 and signal 1 (OKT3) to thetransduced T cells. After 6-8 days, the viability and absolute countwere assessed, and live T cells were rechallenged for an additional weekwith fresh LoVo.OKT3.GFP tumor cells as described above. At the end ofthe long-term coculture, the viability and absolute count were measured,and the fold expansion was calculated. Data shown as mean+/−SEM of n≤3donors analysed in triplicates.

FIGS. 29A-29C depict exhaustion profiles of transduced T cells withCD28.CD40 mutants CoStAR constructs after tumor challenge. Cells ofthree donors were activated with Dynabeads and transduced with CTP194,CTP195, CTP196, CTP197, CTP198, CTP199, CTP200 or mock transduced. Cellswere enriched for CD34 marker expression, expanded following the rapidexpansion protocol (REP) and frozen for subsequent experiments. Afterthaw, cells were rested for 3-4 days in complete RPMI supplemented withIL-2. The viability and absolute count were assessed after overnightIL-2 starvation using DRAQ-7 (1:200) by flow cytometry (Novocyte) anddata were analysed using the NovoExpress 1.5.0 software. Transduced Tcells were cocultured in absence of IL-2 for 6-8 days with LoVo.OKT3.GFPtumor cells at 8:1 effector to target ratio, changing half of theculture medium every 3-4 days. LoVo.OKT3.GFP naturally expresses CEA andPD-L1 on their surface, conferring both signal 2 and signal 1 (OKT3) tothe transduced T cells. After 6-8 days, the viability and the absolutecount were assessed, and live T cells were rechallenged for anadditional week with fresh LoVo.OKT3.GFP tumor cells as described above.Exhaustion profiles (LAG-3 (FIG. 29A), PD-1 (FIG. 29B), TIM-3 (FIG.29C)) of transduced (CD34+(grey)) or non-transduced (CD34− (white)) CD4(upper panels) and CD8 (lower panels) T cells were assessed by flowcytometry and shown as mean+/−SD of n≤3 donors.

FIGS. 30A-30B depict CD28 mutants and IgG4 CD40 CoStAR transduced Tcells secreting higher amount of IL-2 and IFN-γ following activationcompared to mock transduced T cells. (FIG. 30A) Cells of three donorswere activated with Dynabeads and transduced with CTP194, CTP201,CTP202, CTP203 or mock transduced. Cells were enriched for CD34 markerexpression, expanded following the rapid expansion protocol (REP) andfrozen for subsequent experiments. After thaw, cells were rested for 3-4days in complete RPMI supplemented with IL-2 and their transduction ratewas determined looking at the CD34 marker gene expression (A, lowerpanel). (FIG. 30B) The viability and absolute count were assessed afterovernight IL-2 starvation using DRAQ-7 (1:200) by flow cytometry(Novocyte) and data were analysed using the NovoExpress 1.5.0 software.Transduced T cells were cocultured in absence of IL-2 with LoVo(CCL-229™ from ATCC) or LoVo.OKT3.GFP tumor cells at 8:1 effector totarget ratio. After 24 hours, supernatants were collected and frozen.LoVo and LoVo.OKT3.GFP naturally express CEA and PD-L1 on their surface,conferring signal 2 through the CoStAR alone (LoVo) or associated withsignal 1 (LoVo.OKT3.GFP) to the transduced T cells. Cocultures wereperformed in triplicates and corresponding negative (T cells alone,tumor cells alone) and positive (PMA+ionomycin) controls were includedin the experiment. After thaw, secreted IL-2 and IFN-γ were detected byELISA and the absorbance was measured using the FLUOstar Omegamicroplate reader and subsequently analysed with the Omega MARS 3.42 R5software. Each dot represents the mean of triplicates for one donor.Note that negative controls (T cells alone, tumor cells alone) were allbelow the detection range (#).

FIG. 31 depicts the critical role of CD28 PYAP and YMNM motifs in thelong term survival and proliferation of CD40 CoStAR transduced T cellswhen cocultured with LoVo.OKT3. Cells of three donors were activatedwith Dynabeads and transduced with CTP194, CTP201, CTP202, CTP203 ormock transduced. Cells were enriched for CD34 marker expression,expanded following the rapid expansion protocol (REP) and frozen forsubsequent experiments. After thaw, cells were rested for 3-4 days incomplete RPMI supplemented with IL-2. The viability and absolute countwere assessed after overnight IL-2 starvation using DRAQ-7 (1:200) byflow cytometry (Novocyte) and data were analysed using the NovoExpress1.5.0 software. Transduced T cells were cocultured in absence of IL-2for 6-8 days with LoVo.OKT3.GFP tumor cells at 8:1 effector to targetratio, changing half of the culture medium every 3-4 days. LoVo.OKT3.GFPnaturally expresses CEA and PD-L1 on their surface, conferring bothsignal 2 and signal 1 (OKT3) to the transduced T cells. After 6-8 days,the viability and absolute count were assessed, and live T cells wererechallenged for an additional week with fresh LoVo.OKT3.GFP tumor cellsas described above. At the end of the long-term coculture, the viabilityand absolute count were measured, and the fold expansion was calculated.Data shown as mean+/−SEM of n≤3 donors analysed in triplicates.

FIGS. 32A-32C depict exhaustion profiles of transduced T cells with CD28mutant CD40 CoStAR constructs after tumor challenge. Cells of threedonors were activated with Dynabeads and transduced (spinoculation, MOI5) with CTP194, CTP201, CTP202, CTP203 or mock transduced. Cells wereenriched for CD34 marker expression, expanded following the rapidexpansion protocol (REP) and frozen for subsequent experiments. Afterthaw, cells were rested for 3-4 days in complete RPMI supplemented withIL-2. The viability and absolute count were assessed after overnightIL-2 starvation using DRAQ-7 (1:200) by flow cytometry (Novocyte) anddata were analysed using the NovoExpress 1.5.0 software. Transduced Tcells were cocultured in absence of IL-2 for 6-8 days with LoVo.OKT3.GFPtumor cells at 8:1 effector to target ratio, changing half of theculture medium every 3-4 days. LoVo.OKT3.GFP naturally expresses CEA andPD-L1 on their surface, conferring both signal 2 and signal 1 (OKT3) tothe transduced T cells. After 6-8 days, the viability and the absolutecount were assessed, and live T cells were rechallenged for anadditional week with fresh LoVo.OKT3.GFP tumor cells as described above.Exhaustion profiles (LAG-3 (FIG. 32A), PD-1 (FIG. 32B), TIM-3 (FIG.32C)) of transduced (CD34+(grey)) or non-transduced (CD34− (white)) CD4(upper panels) and CD8 (lower panels) T cells were assessed by flowcytometry and shown as mean+/−SD of n≤3 donors.

FIG. 33 depicts generation of transduced T cells from four healthydonors following CD34 enrichment and expansion. T cells of 4 healthydonors (NBC360, NBC362, NBC358, NBC361) were activated with Dynabeadsand transduced with CTP188, CTP189, CTP190, CTP191, CTP192, CTP193,CTP194, CTP195, CTP196, CTP197, CTP198, CTP199, CTP200, CTP201, CTP202,CTP203, CTP204 or mock transduced. Cells were then magnetically enrichedfor their CD34 expression and expanded following the rapid expansionprotocol (REP). Viability of each sample 10-11 days after REP wasassessed by flow cytometry (Novocyte). Data were analysed withNovoExpress 1.5.0 software. Each dot within the same donor represents adifferent construct.

FIGS. 34A-34D depict expression of activation markers and cytokineproduction of non-transduced (NTD) or anti-FOLR1 CoStAR modified T cells(CoStAR) from 3 healthy donors co-cultured overnight with Ba/F3 targets.Expression of activation markers 4-1BB and CD69 (FIG. 34A) andproduction of IL-2 and IFNγ (FIG. 34B) was determined. CoStAR engagementenhances cytokine secretion. (FIG. 34C) Non-transduced and CoStARcytotoxicity is comparable. Tumor counts of Ba/F3 targets were assessedby flow cytometry after overnight coculture with non-transduced (NTD)and CoStAR T cells. (FIG. 34D) CoStAR engagement enhances both CD4 andCD8 T cell proliferation. NTD and CoStAR T cell counts as well asproliferation were assessed by flow cytometry after overnight or 5-daycoculture with Ba/F3 targets.

FIGS. 35A-35D depicts expression of activation markers of anti-FOLR1CoStAR modified T cells (CoStAR) from 3 healthy donors preincubated withsoluble folate receptor (sFOLR1) followed by co-culture overnight withBa/F3 targets. X-axis shows sFOLR1 as ng/mL. In each group, bars 1˜4 arenon-transduced, bars 5-8 are CoStAR transduced. Soluble FOLR1 does notimpact upregulation of activation markers on CoStAR T cells. Expressionof activation markers 4-1BB (FIG. 35A) and CD69 (FIG. 35B) wasdetermined. (FIG. 35C) sFOLR1 does not impact cytoxicity of CoStAR Tcells. Tumor counts of Ba/F3 targets were assessed by flow cytometryafter overnight coculture with non-transduced (NTD) or CoStAR transducedT cells pre-incubated with increasing concentrations of sFOLR. (FIG.35D) sFOLR1 does not impact cytokine secretion. IL-2 production is shownin anti-FOLR1 CoStAR modified T cells preincubated with soluble folatereceptor (sFOLR) followed by co-culture overnight with Ba/F3 targets.

FIGS. 36A-36D depict FOLR1 CoStAR requires signal 1 to function. (FIGS.36A and 36B) Expression of activation markers (FIG. 36A) and cytokineproduction (FIG. 36B) respectively, from non-transduced (NTD) andanti-FOLR1 CoStAR modified T cells (CoStAR) from 3 healthy donorsco-cultured overnight with Ba/F3 targets. (FIG. 36A) There is minimalupregulation of activation markers with signal 2 only. (FIG. 36B) Nocytokine secretion was observed with signal 2 only. (FIG. 36C) Tumorcounts of Ba/F3 targets assessed by flow cytometry after overnightcoculture with NTD and CoStAR T cells. No cytoxicity was observed withsignal 2 only. (FIG. 36D) NTD and CoStAR T cell counts were assessed byflow cytometry after overnight or 5-day coculture with Ba/F3 targets.Proliferation was not observed with signal 2 only.

FIGS. 37A-37C depict TIL function with CoStAR in ovarian cancer. (FIG.37A) TIL from 6 ovarian tumors were liberated by digestion and culturedin 3000 U IL-2. Transduction with a 3^(rd) generation lentiviral vectorencoding a CoStAR molecule with and scFv targeting human FOLR1, linker,full length CD28 fused to truncated CD40 cytoplasmic domain was carriedout at an MOI of 5, both 48 h and 72 h after tumor digestion. Rapidexpansion protocol was carried out on days 12-23. (FIG. 37B) Flowcytometric analysis was used to determine the frequency of CD4 and CD8T-cells expressing the CoStAR molecule using an anti-idiotype antibodyfor surface detection. (FIG. 37C) Flow cytometric analysis was used todetermine the frequency of cells expressing TCRαβ and TCRγδ by flowcytometric surface staining. Mock—untransduced cells. CoStAR^(−/+):cells negative or positive for CoStAR molecule in the treated cellpopulation as determined by flow cytometry gating.

FIGS. 38A-38B depict TIL function with CoStAR in ovarian cancer. (FIG.38A) CoStAR modified TIL from 5 ovarian tumors were co-cultured withautologous digest overnight in the presence of brefeldin A. Thefrequency of cells expressing IL-2 or TNFα was assessed the followingday by flow cytometry. The frequency of TIL reacting to autologousdigest is enhanced by the CoStAR molecule. NTD: untransduced cells.CoStAR^(−/+:) cells negative or positive for CoStAR molecule in thetreated cell population as determined by flow cytometry gating. (FIG.38B) CoStAR modified TIL from 5 ovarian tumors were co-cultured withautologous digest and supernatant assessed for cytokine release. CoStARmodified cells had increased effector functions as demonstrated byincreased IFNγ, TNFα and IL-13 release. Maximal levels of thesemolecules was similar in response to stimulation with PMA (Phorbol12-myristate 13-acetate) and ionomycin.

FIGS. 39A-39C depict CoStAR TIL retain robust effector functions andretain a requirement for signal 1 and 2. (FIG. 39A) CoStAR modified TILfrom 5 ovarian tumors were co-cultured with BA/F3 cells or BA/F3 cellsengineered to express OKT3, FOLR1 or both. Cytokine secretion ofnon-modified and CoStAR modified TIL was equivalent when co-culturedwith non-modified BA/F3 or BA/F3 expressing OKT3 alone or FOLR1 alone.CoStAR modified TIL secreted increased levels of cytokines whenco-cultured with BA/F3 modified to express both FOLR1 and OKT3. (FIG.39B) CoStAR modified TIL from 5 ovarian tumors were co-cultured withBA/F3 cells or BA/F3 cells engineered to express OKT3, FOLR1 or both.Cytotoxicity towards BA/F3 target cells was assessed via cell counts,determined by flow cytometric analysis of mouse CD45. Non-modified andCoStAR modified cells kill cells expressing OKT3 equivalently. CoStARmodified TILs do not kill BA/F3 cells expressing FOLR1 alone. (FIG. 39C)Mock or CoStAR modified TIL from 3 ovarian cancer patients wereco-cultured with autologous tumor in the presence of no blocking, MHCI,MHC II or MHC I+MHC II blocking or antibodies or isotype control.Supernatant was assessed for the level of IFNγ release. Normalized tolevels of release without antibody, IFNγ levels are similarly reducedmock and CoStAR modified TIL relative to no antibody or isotype controlconditions, showing that activity is led by endogenous TCR-MHC peptideinteractions.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that costimulatory receptors comprising aCD40 signaling domain display novel and improved activity profiles. Theactivity profiles can be modulated by selecting an intracellular domainof a receptor protein for joining to the CD40 signaling domain and/or byselecting elements of the CD40 signaling domains to join to theintracellular domain of a receptor protein. Provided herein arerecombinant costimulatory antigen receptors (CoStARs) comprising: (i) adisease- or tumor-associated antigen binding domain, (ii) a firstintracellular segment comprising an intracellular signaling domain of areceptor protein, and (iii) a second intracellular signaling domain of aCD40 receptor protein or signal transducing fragment thereof.Optionally, the CoStAR comprises an extracellular segment of astimulatory receptor protein. In certain embodiments, the extracellularsegment of the stimulatory receptor protein is capable of bindingligand. In certain embodiments, the extracellular segment of astimulatory receptor protein is truncated and does not bind ligand. Incertain embodiments the extracellular segment of the stimulatoryreceptor protein operates as an adjustable length spacer allowing thedisease- or tumor-associated antigen binding domain to be located awayfrom the surface of the cell in which it is expressed for example toform a more optimal immune synapse. In certain embodiments, theextracellular segment of a stimulatory receptor protein and the firstintracellular segment comprise segments of the same receptor protein. Incertain embodiments, the extracellular segment and the firstintracellular segment comprise segments of different receptor proteins.The CoStARs comprise an intervening transmembrane domain between thedisease or tumor antigen binding domain and the first intracellulardomain. When an extracellular segment of a stimulatory receptor proteinis present, the transmembrane domain is intervening between theextracellular segment and the first intracellular signaling domain.

As used herein, “full length protein” or “full length receptor” refersto a receptor protein, such as, for example, a CD28 receptor protein.The term “full length” encompasses receptor proteins lacking up to about5 or up to 10 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids, at the N-terminal of the mature receptor protein once itssignal peptide has been cleaved. For instance, while a specific cleavagesite of a receptors N-terminal signal peptide may be defined,variability in exact point of cleavage has been observed. The term “fulllength” does not imply presence or absence of amino acids of thereceptors N-terminal signal peptide. In one embodiment, the term “fulllength” (e.g. a full length CD28 or a full length CD40 intracellulardomain, according to certain aspects of the invention) encompassesmature receptor proteins (e.g. CD28 according to certain aspects of theinvention) lacking the N terminal signal peptide lacking up to about 5,for example 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminal ofthe mature receptor protein once its signal peptide has been cleaved. Asmentioned above, a “full length” CD28 receptor or other receptor ortumor antigen binding domain according to the various aspects of theinvention does not include the signal peptide and may lack up to about5, for example 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminalof the mature receptor protein (e.g. N terminal residues N, K, I, Land/or V). This is shown in the exemplary fusions, e.g. SEQ ID Nos. 4-12(note that these may lack up to about 5, for example 1, 2, 3, 4, 5, orup to 10 amino acids at the N-terminal of the mature receptor protein asshown in the boxed region).

CoStARs have modular form and can be constructed to compriseextracellular, transmembrane and intracellular domains obtained from aone or more proteins, along with the scFv obtained from an antibody thatbinds to a disease-associated antigen, for example, a tumor associatedantigen.

According to the invention, in one embodiment, a CoStAR comprises adisease-associated, for example a tumor-associated, antigen receptor,such as but not limited to a tumor-associated antigen specific scFv, anda primary costimulatory receptor protein that is capable of binding toits cognate ligand and providing an intracellular signal. In certainembodiments, the primary costimulatory receptor can be less than a fulllength protein but is sufficient to bind cognate ligand and transduce asignal. In certain embodiments, the primary costimulatory receptordomain is full length, such as but not limited to, full length CD28.Thus, both the antigen specific binding domain and the ligand specificreceptor are capable of binding cognate antigen and ligand respectively.The amino acid sequences provided herein provide embodiments of severalCoStAR constructs. These include CoStARs constructs that comprise anantigen binding domain, an optional spacer, an optional costimulatoryreceptor protein comprising an extracellular ligand binding segment orfragment thereof and intracellular CD40 signaling domain. In anotherembodiment, a CoStAR comprises an antigen binding domain, an optionalspacer, an extracellular ligand-binding portion of a costimulatoryreceptor protein, a transmembrane domain, and an intracellular signalingdomain of a selected costimulatory receptor protein and intracellularCD40 signaling domain. In certain embodiments, the extracellularligand-binding portion comprises a CD28 truncation, for example, aC-terminal CD28 truncation after amino acids IEV, and is followed by anintracellular signaling domain. In certain embodiments, theintracellular signaling domain is from CD40. The transmembrane domainseparating the extracellular ligand-binding and intracellular signalingdomains can be from, with limitation, CD28, CD40. In furtherembodiments, CoStARs can comprise additional costimulatory domains, forexample a third, intracellular costimulatory signaling domain and inthis respect may be similar to certain chimeric antigen receptors(CARs), which have been classified into first (CD3ζ only), second (onecostimulatory domain+CD3ζ), or third generation (more than onecostimulatory domain+CD3ζ).

Costimulatory receptor proteins useful in CoStARs of the inventioninclude, without limitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38,CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134(OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357(GITR), or EphB6, which in their natural form comprise extracellularligand binding domains and intracellular signal transducing domains. Forexample, CD2 is characterized as a cell adhesion molecule found on thesurface of T cells and is capable of initiating intracellular signalsnecessary for T cell activation. CD27 is characterized as a type IItransmembrane glycoprotein belonging to the TNFR superfamily (TNFRSF)whose expression on B cells is induced by antigen-receptor activation inB cells. CD28 is one of the proteins on T cells and is the receptor forCD80 (B7.1) and CD86 (B7.2) ligands on antigen-presenting cells. CD137(4-1BB) ligand is found on most leukocytes and on some non-immune cells.OX40 ligand is expressed on many antigen-presenting cells such as DC2s(dendritic cells), macrophages, and B lymphocytes. In one embodiment,the costimulatory receptor protein is full length CD28 as definedherein.

CD40 is a member of the tumor necrosis factor receptor (TNFR)superfamily and several isoforms are generated by alternative splicing.Its ligand, CD154 (also called CD40L) is a protein that is primarilyexpressed on activated T cells. For reference, the human CD40 isoform 1protein sequence is set forth in GenBank accession No. NP_001241.1,including signal peptide (amino acids 1-20), transmembrane domain (aminoacids 194-215), and cytoplasmic domain (amino acids 216-277)(SEQ IDNO:22). CD40 receptor signaling involves adaptor proteins including butnot limited to TNF receptor-associated factors (TRAF), and the CD40cytoplasmic domain comprises signaling components, including amino acidsequences fitting an SH3 motif (KPTNKAPH or PTNKAPHP or PTNKAPH), TRAF2motif (PKQE, PKQET, PVQE, PVQET, SVQE, SVQET), TRAF6 motif (QEPQEINF orQEPQEINFP) and PKA motif (KKPTNKA, SRISVQE). The invention furtherincludes engineered signaling domains, such as engineered CD40 signalingdomains, comprising TRAF-binding amino acid sequences. Engineeredsignaling domains that bind to TRAF1, TRAF2, TRAF3, and TRAF5 maycomprise the major consensus sequence (P/S/A/T)X(Q/E)E or minorconsensus sequence PXQXXD and can be identified in or obtained from,without limitation, TNFR family members such as CD30, Ox40, 4-1BB, andthe EBV oncoprotein LMP1. (See, e.g., Ye, H et al., The Structural Basisfor the Recognition of Diverse Receptor Sequences by TRAF2. MolecularCell, 1999; 4(3):321-30. doi: 10.1016/S1097-2765(00)80334-2; Park H H,Structure of TRAF Family: Current Understanding of Receptor Recognition.Front. Immunol. 2018; 9:1999. doi: 10.3389/fimmu.2018.01999; Chung, J.Y. et al., All TRAFs are not created equal: common and distinctmolecular mechanisms of TRAF-mediated signal transduction. Journal ofCell Science 2002; 115:679-688).

Examples disclosed herein demonstrate operation of CD40 as acostimulatory signaling domain in a CoStAR and further that cytokine andchemokine expression profiles are altered by signaling domain selection.In certain embodiments, the costimulatory CD40 signaling domain of aCoStAR promotes pro-inflammatory cytokines (e.g., IL-2, TNFα). Incertain embodiments, the costimulatory CD40 signaling domain of a CoStARreduces immunosuppressive cytokines (e.g., IL-5, IL-10). Costimulatoryactivity of a CD40 signaling domain or fragment can be observed incombination with a first receptor signaling domain such as but notlimited to CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46,CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB),CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), or EphB6, ascompared to activity of the first receptor signaling domain without theCD40 signaling domain or fragment. In this regard, the CD40 signalingdomains of the invention, including signaling fragments comprisingparticular factor binding sites or wherein particular factor bindingsites are mutated, in combination with a costimulatory first signalingdomain, are capable of promoting or suppressing relative expression ofparticular cytokines and/or chemokines as compared to the firstsignaling domain alone. activity of a costimulatory signaling domain.(See, e.g., Ahonen, C L et al., The CD40-TRAF6 axis controls affinitymaturation and the generation of long-lived plasma cells. Nat Immunol.2002; 3: 451-456; Mackey M F et al., Distinct contributions of differentCD40 TRAF binding sites to CD154-induced dendritic cell maturation andIL-12 secretion. Eur J Immunol. 2003; 33: 779-789; Mukundan Let al., TNFreceptor-associated factor 6 is an essential mediator of CD40-activatedproinflammatory pathways in monocytes and macrophages. J Immunol. 2005;174: 1081-1090.

In certain embodiments, a CoStAR of the invention comprisessubstantially all of a CD40 costimulatory domain. In certainembodiments, a CoStAR of the invention comprises two or more CD40costimulatory domains. In certain embodiments, a CoStAR of the inventioncomprises a CD40 costimulatory domain signaling component or motif,including but not limited to an SH3 motif (KPTNKAPH), TRAF2 motif (PKQE,PVQE, SVQE), TRAF3 motif, TRAF6 motif (QEPQEINFP) or PKA motif (KKPTNKA,SRISVQE) as well as two or more, or three or more, or four or more suchcomponents of motifs, which can be in multiple copies and arranged inany order. In certain embodiments, a CoStAR of the invention comprises aCD40 costimulatory domain and a CD40 costimulatory domain signalingcomponent or motif. In certain embodiments, the SH3 motif, TRAF2 motif,and TRAF6 motif are sufficient to modulate pro-inflammatory and/orimmunosuppressive cytokines. In certain embodiments, adding tandemcopies of those motifs and/or mutating certain motifs amplifies theseeffects.

In certain embodiments, selection of one or more costimulatory domainsignaling component or motif is guided by the cell in which the CoStARis to be expressed and/or a desired costimulatory activity more closelyidentified with a signaling component or motif, or avoidance of acostimulatory activity more closely identified with a signalingcomponent or motif.

In certain embodiments, a CoStAR signaling domain comprises, in additionto a CD40 costimulatory domain or signaling component or motif thereof,or two or more such domains or components or motifs or combinationsthereof, an additional full length costimulatory domain or signalingcomponent thereof from, without limitation, CD2, CD9, CD26, CD27, CD28,CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99,CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278(ICOS), CD357 (GITR), or EphB6,

For reference, the human CD28 protein sequence is set forth in GenBankaccession No. NP_006130.1, including signal peptide (amino acids 1-18),extracellular domain (amino acids 19-152), transmembrane domain (aminoacids 153-179) and cytoplasmic domain (amino acids 180-200). Theextracellular domain includes an immunoglobulin type domain (amino acids21-136) which contains amino acids with compose the antigen binding siteand amino acids that form the homodimer interface. The extracellulardomain includes several asparagine residues which may be glycosylated,and the intracellular domain comprises serine and tyrosine residues,which may be phosphorylated.

For reference, the human CD8 alpha chain protein sequence is set forthby GenBank accession No. NP_001139345.1, including signal peptide (aminoacids 1-21), extracellular domain (amino acids 22-182), transmembranedomain (amino acids 183-203), and cytoplasmic domain (amino acids204-235). The extracellular domain includes an immunoglobulin typedomain (amino acids 28-128) which contains amino acids with compose theantigen binding site and amino acids that form the homodimer interface.The extracellular domain includes several asparagine residues which maybe glycosylated, and the intracellular domain comprises serine andtyrosine residues, which may be phosphorylated.

For reference, the human IgG4 constant region sequence is set forth inUniProtKB/Swiss-Prot: accession No. P01861.1, including CH1 (amino acids1-98), hinge (amino acids 99-110), CH2 (amino acids 111-220), CH3 (aminoacids 221-327). The CH2 region includes asparagine at amino acid 177,which is the glycosylated and associated with Fc receptor andantibody-dependent cell-mediated cytotoxicity (ADCC).

For reference, the protein sequence of human CD137 (4-1BB), another TNFRsuperfamily member, is set forth by GenBank accession No. NP_001552.2,including signal peptide (amino acids 1-23), extracellular domain (aminoacids 24-186), transmembrane domain (amino acids 187-213), andcytoplasmic domain (amino acids 214-255). Binding of CD137L ligandtrimers expressed on antigen presenting cells to CD137 leads to receptortrimerization and activation of signaling cascades involved in T cellreactivity and survival (Li et al., Limited Cross-Linking of 4-1BB by4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab. CellReports 2018; 25:909-920). Coimmunoprecipitation of CD137 with thesignaling adaptors TRAF-2 and TRAF-1 and the structural basis for theinteractions has been reported (Ye, H et al., Molecular Cell, 1999;4(3):321-30).

For reference, the human CD134 (OX40) protein sequence is set forth byGenBank accession No. NP_003318.1, including signal peptide (amino acids1-28), extracellular domain (amino acids 29-214), transmembrane domain(amino acids 215-235), and cytoplasmic domain (amino acids 236-277).This receptor has been shown to activate NF-kappaB through itsinteraction with adaptor proteins TRAF2 and TRAF5 and studies suggestthat this receptor promotes expression of apoptosis inhibitors BCL2 andBCL21L1/BCL2-XL.

The human T-cell surface antigen CD2 has at least two isoforms. Forreference, the human CD2 isoform1 protein sequence is set forth byNP_001315538.1, including signal peptide (amino acids 1-24),extracellular domain (amino acids 25-235), transmembrane domain (aminoacids 236-261), and cytoplasmic domain (amino acids 262-377). The humanCD2 isoform2 protein sequence is set forth by NP_001758.2

For reference, the human CD357 (GITR) isoform-1 protein sequence is setforth by GenBank accession No. NP_004186.1, including signal peptide(amino acids 1-25), extracellular domain (amino acids 26-162),transmembrane domain (amino acids 163-183), and cytoplasmic domain(amino acids 184-241).

For reference, the human CD29 (beta1 integrin) protein sequence is setforth by GenBank accession No. NP_596867, including signal peptide(amino acids 1-20), extracellular domain (amino acids 21-728),transmembrane domain (amino acids 729-751), and cytoplasmic domain(amino acids 752-798).

The human CD150 (SLAM) protein sequence has at several isoforms. Inaddition to the transmembrane form of CD150 (mCD150), cells ofhematopoietic lineage express mRNA encoding the secreted form of CD150(sCD150), which lacks the entire transmembrane region of 30 amino acids.For reference, human SLAM isoform b is set forth by GenBank accessionNo. NP_003028.1, including signal peptide (amino acids 1-20),extracellular domain (amino acids 21-237), transmembrane domain (aminoacids 238-258), and cytoplasmic domain (amino acids 259-335). Human SLAMisoform a is set forth by GenBank accession No. NP_001317683.1.

In embodiments of the invention, a CoStAR may be expressed alone underthe control of a promoter in a therapeutic population of cells that havetherapeutic activity, for example, Tumor Infiltrating Lymphocytes(TILs). Alternatively, the CoStAR may be expressed along with atherapeutic transgene such as a chimeric antigen receptor (CAR) and/orT-cell Receptor (TCR), for example as described in SEQ ID NOS:67-79(note that may lack up to about 5, for example 1, 2, 3, 4, 5, or up to10 amino acids at the N-terminal of the mature receptor protein). Thus,in one aspect, the invention also relates to CoStAR constructs having asequence as shown in any of SEQ ID NOS:67-79, including one of thesesequences which lacks up to about 5, for example 1, 2, 3, 4, 5, or up to10 amino acids at the N-terminal of the mature receptor protein).Suitable TCRs and CARs are well known in the literature, for exampleHLA-A*02-NYESO-1 specific TCRs (Rapoport et al. Nat Med 2015) oranti-CD19scFv.CD3ζ fusion CARs (Kochenderfer et al. J Clin Oncol 2015)which have been successfully used to treat Myeloma or B-cellmalignancies respectively. The CoStARs described herein may be expressedwith any known CAR or TCR thus providing the cell with a regulatablegrowth switch to allow cell expansion in-vitro or in-vivo, and aconventional activation mechanism in the form of the TCR or CAR foranti-cancer activity. Thus the invention provides a cell for use inadoptive cell therapy comprising a CoStAR as described herein and a TCRand/or CAR that specifically binds to a tumor associated antigen. Anexemplary CoStAR comprising CD28 includes an extracellular antigenbinding domain and an extracellular, transmembrane and intracellularsignaling domain.

The term “antigen binding domain” as used herein refers to an antibodyfragment including, but not limited to, a diabody, a Fab, a Fab′, aF(ab′) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a(dsFv)2, a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody(ds diabody), a single-chain antibody molecule (scFv), an scFv dimer(bivalent diabody), a multispecific antibody formed from a portion of anantibody comprising one or more CDRs, a camelized single domainantibody, a nanobody, a domain antibody, a bivalent domain antibody, orany other antibody fragment that binds to an antigen but does notcomprise a complete antibody structure. An antigen binding domain iscapable of binding to the same antigen to which the parent antibody or aparent antibody fragment (e.g., a parent scFv) binds. In someembodiments, an antigen-binding fragment may comprise one or morecomplementarity determining regions (CDRs) from a particular humanantibody grafted to frameworks (FRs) from one or more different humanantibodies.

The antigen binding domain can be made specific for anydisease-associated antigen, including but not limited totumor-associated antigens (TAAs) and infectious disease-associatedantigens. In certain embodiments, the ligand binding domain isbispecific. Antigens have been identified in most of the human cancers,including Burkitt lymphoma, neuroblastoma, melanoma, osteosarcoma, renalcell carcinoma, breast cancer, prostate cancer, lung carcinoma, andcolon cancer. TAA's include, without limitation, CD19, CD20, CD22, CD24,CD33, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα),mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP,SM5-1, MICA, MICB, ULBP and HER-2. TAAs further include neoantigens,peptide/MHC complexes, and HSP/peptide complexes.

In certain embodiments, the antigen binding domain comprises a T-cellreceptor or binding fragment thereof that binds to a defined tumorspecific peptide-MHC complex. The term “T cell receptor,” or “TCR,”refers to a heterodimeric receptor composed of αβ or γδ chains that pairon the surface of a T cell. Each α, β, γ, and δ chain is composed of twoIg-like domains: a variable domain (V) that confers antigen recognitionthrough the complementarity determining regions (CDR), followed by aconstant domain (C) that is anchored to cell membrane by a connectingpeptide and a transmembrane (TM) region. The TM region associates withthe invariant subunits of the CD3 signaling apparatus. Each of the Vdomains has three CDRs. These CDRs interact with a complex between anantigenic peptide bound to a protein encoded by the majorhistocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature,334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy(2012), xix, 868 p.).

In certain embodiments, the antigen binding domain comprises a naturalligand of a tumor expressed protein or tumor-binding fragment thereof. Anon-limiting example is PD1 which binds to PDL1. Another example is thetransferrin receptor 1 (TfR1), also known as CD71, a homodimeric proteinthat is a key regulator of cellular iron homeostasis and proliferation.Although TfR1 is expressed at a low level in a broad variety of cells,it is expressed at higher levels in rapidly proliferating cells,including malignant cells in which overexpression has been associatedwith poor prognosis. In an embodiment of the invention, the antigenbinding domain comprises transferrin or a transferrin receptor-bindingfragment thereof.

In certain embodiments, the antigen binding domain is specific to adefined tumor associated antigen, such as but not limited to FRα, CEA,5T4, CA125, SM5-1 or CD71. In certain embodiments, the tumor associatedantigen can be a tumor-specific peptide-MHC complex. In certain suchembodiments, the peptide is a neoantigen. In other embodiments, thetumor associated antigen it a peptide-heat shock protein complex.

In various embodiments, the invention provides a CoStAR which comprises

i. an scFv that binds to carcinoembryonic antigen (CEA), a spacer andtransmembrane sequence of CD28, a CD28 signaling domain, and a CD40signaling domain.

ii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, and a CD40 signaling domain.

iii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a CD137 signaling domain, and a CD40 signaling domain.

iv. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a CD134 signaling domain, and a CD40 signaling domain.

v. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a CD2 signaling domain, and a CD40 signaling domain.

vi. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a GITR signaling domain, and a CD40 signaling domain.

vii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a CD29 signaling domain, and a CD40 signaling domain.

viii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD28, a CD150 signaling domain, and a CD40 signaling domain.

ix. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD28 signaling domain, and a CD40 signaling domain.

x. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, and a CD40 signaling domain.

xi. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD137 signaling domain, and a CD40 signaling domain.

xii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD134 signaling domain, and a CD40 signaling domain.

xiii. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD2 signaling domain, and a CD40 signaling domain.

xiv. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a GITR signaling domain, and a CD40 signaling domain.

xv. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD29 signaling domain, and a CD40 signaling domain.

xvi. an scFv that binds to CEA, a spacer and transmembrane sequence ofCD8, a CD150 signaling domain, and a CD40 signaling domain.

xvii. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD28 signaling domain, and aCD40 signaling domain.

xviii. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, and a CD40 signaling domain.

xix. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD137 signaling domain, and aCD40 signaling domain.

xx. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD134 signaling domain, and aCD40 signaling domain.

xxi. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD2 signaling domain, and aCD40 signaling domain.

xxii. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a GITR signaling domain, and aCD40 signaling domain.

xxiii. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD29 signaling domain, and aCD40 signaling domain.

xxiv. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD150 signaling domain, and aCD40 signaling domain.

xxv. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a first CD40 signaling domainand a second CD40 signaling domain

xxvi. an scFv that binds to CEA, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a first CD40 signaling domainand a second mutated CD40 signaling domain

xxvii. a binding domain that binds to PDL1, a short spacer andtransmembrane sequence of CD28, a CD28 signaling domain, and a CD40signaling domain.

xxviii. a binding domain that binds to PDL1, a short spacer andtransmembrane sequence of CD28, and a CD40 signaling domain.

xxix. an binding domain that binds to CD155, CD112, or CD113, a CD28transmembrane domain, a CD28 signaling domain, and a CD40 signalingdomain.

xxx. a binding domain that binds to CD155, CD112, or CD113, a CD28transmembrane domain, and a CD40 signaling domain.

xxxi. an scFv that binds to CEA, a binding domain that binds to PDL1, ashort spacer and transmembrane sequence of CD28, a CD28 signalingdomain, and a CD40 signaling domain.

xxxii. an scFv that binds to CEA, a binding domain that binds to PDL1, ashort spacer and transmembrane sequence of CD28, and a CD40 signalingdomain.

xxxiii. an scFv that binds to CEA, a binding domain that binds to CD155,CD112, or CD113, a short spacer and transmembrane sequence of CD28, aCD28 signaling domain, and a CD40 signaling domain.

xxxiv. an scFv that binds to CEA, a binding domain that binds to CD155,CD112, or CD113, a short spacer and transmembrane sequence of CD28, anda CD40 signaling domain.

In various embodiments, the invention provides a CoStAR which comprisesthe spacer, transmembrane, and signaling domain structure of any one ofi-xxxiv and binds to FOLR1.

In various embodiments, the invention provides a CoStAR which comprises

i. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a CD28 signaling domain, and a CD40 signaling domain.

ii. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, and a CD40 signaling domain.

iii. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a CD137 signaling domain, and a CD40 signaling domain.

iv. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a CD134 signaling domain, and a CD40 signaling domain.

v. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a CD2 signaling domain, and a CD40 signaling domain.

vi. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a GITR signaling domain, and a CD40 signaling domain.

vii. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD28, a CD29 signaling domain, and a CD40 signaling domain.

viii. an scFv that binds to FOLR1, a spacer and transmembrane sequenceof CD28, a CD150 signaling domain, and a CD40 signaling domain.

ix. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a CD28 signaling domain, and a CD40 signaling domain.

x. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, and a CD40 signaling domain.

xi. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a CD137 signaling domain, and a CD40 signaling domain.

xii. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a CD134 signaling domain, and a CD40 signaling domain.

xiii. an scFv that binds to FOLR1, a spacer and transmembrane sequenceof CD8, a CD2 signaling domain, and a CD40 signaling domain.

xiv. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a GITR signaling domain, and a CD40 signaling domain.

xv. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a CD29 signaling domain, and a CD40 signaling domain.

xvi. an scFv that binds to FOLR1, a spacer and transmembrane sequence ofCD8, a CD150 signaling domain, and a CD40 signaling domain.

xvii. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD28 signaling domain, and aCD40 signaling domain.

xviii. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, and a CD40 signaling domain.

xix. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD137 signaling domain, and aCD40 signaling domain.

xx. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD134 signaling domain, and aCD40 signaling domain.

xxi. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD2 signaling domain, and aCD40 signaling domain.

xxii. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a GITR signaling domain, and aCD40 signaling domain.

xxiii. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD29 signaling domain, and aCD40 signaling domain.

xxiv. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a CD150 signaling domain, and aCD40 signaling domain.

xxv. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a first CD40 signaling domainand a second CD40 signaling domain

xxvi. an scFv that binds to FOLR1, a spacer comprising an IgG4 constantregion and CD28 transmembrane sequence, a first CD40 signaling domainand a second mutated CD40 signaling domain

As use herein, the term “specifically binds” or “is specific for” refersto measurable and reproducible interactions, such as binding between atarget and an antibody or antibody moiety that is determinative of thepresence of the target in the presence of a heterogeneous population ofmolecules, including biological molecules. For example, an antibodymoiety that specifically binds to a target (which can be an epitope) isan antibody moiety that binds the target with greater affinity, avidity,more readily, and/or with greater duration than its bindings to othertargets. In some embodiments, an antibody moiety that specifically bindsto an antigen reacts with one or more antigenic determinants of theantigen (for example a cell surface antigen or a peptide/MHC proteincomplex) with a binding affinity that is at least about 10 times itsbinding affinity for other targets.

Spacer

A CoStAR of the invention optionally comprises a spacer region betweenthe antigen binding domain and the costimulatory receptor. As usedherein, the term “spacer” refers to the extracellular structural regionof a CoStAR that separates the antigen binding domain from the externalligand binding domain of the costimulatory protein. The spacer providesflexibility to access the targeted antigen and receptor ligand. Incertain embodiments long spacers are employed, for example to targetmembrane-proximal epitopes or glycosylated antigens (see Guest R. D. etal. The role of extracellular spacer regions in the optimal design ofchimeric immune receptors: evaluation of four different scFvs andantigens. J. Immunother. 2005; 28:203-211; Wilkie S. et al., Retargetingof human T cells to tumor-associated MUC1: the evolution of a chimericantigen receptor. J. Immunol. 2008; 180:4901-4909). In otherembodiments, CoStARs bear short spacers, for example to target membranedistal epitopes (see Hudecek M. et al., Receptor affinity andextracellular domain modifications affect tumor recognition byROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al., The nonsignalling extracellular spacerdomain of chimeric antigen receptors is decisive for in vivo antitumoractivity. Cancer Immunol. Res. 2015; 3:125-135). In certain embodiments,the spacer comprises all or part of or is derived from an IgG hinge,including but not limited to IgG1, IgG2, or IgG4. By “derived from an Ighinge” is meant a spacer comprising insertions, deletions, or mutationsin an IgG hinge. In certain embodiments, a spacer can comprise all orpart of one or more antibody constant domains, such as but not limitedto CH2 and/or CH3 domains. In certain embodiments, in a spacercomprising all or part of a CH2 domain, the CH2 domain is modified so asnot to bind to an Fc receptor. For example, Fc receptor binding inmyeloid cells has been found to impair CAR T cell functionality. Incertain embodiments, the spacer comprises all or part of an Ig-likehinge from CD28, CD8, or other protein comprising a hinge region. Incertain embodiments of the invention that comprise a spacer, the spaceris from 1 and 50 amino acids in length.

In an non-limiting embodiment, the spacer comprises essentially all ofan extracellular domain, for example a CD28 extracellular domain (i.e.from about amino acid 19, 20, 21, or 22 to about amino acid 152) or anextracellular domain of another protein, including but not limited toanother TNFR superfamily member. In an embodiment, the spacer comprisesa portion of an extracellular domain, for example a portion of a CD28extracellular domain, and may lack all or most of the Ig domain. Inanother embodiment, the spacer includes amino acids of CD28 from about141 to about 152 but not other portions of the CD28 extracellulardomain. In another embodiment, the spacer includes amino acids of CD8from about 128 to about 182 but not other portions of the CD8extracellular domain.

Linker

In certain embodiments, the CoStAR extracellular domain comprises alinker. Linkers comprise short runs of amino acids used to connectdomains, for example a binding domain with a spacer or transmembranedomain. In order for there to be flexibility to bind ligand, a ligandbinding domain will usually be connected to a spacer or a transmembranedomain by flexible linker comprising from about 5 to 25 amino acids,such as, for example, AAAGSGGSG (SEQ ID NO:7), GGGGSGGGGSGGGGS (SEQ IDNO:62). In certain embodiments, a CoStAR comprises a binding domainjoined directly to a transmembrane domain by a linker, and without aspacer. In certain embodiments, a CoStAR comprises a binding domainjoined directly to a transmembrane by a spacer and without a linker,exemplified by SEQ ID NOS:58 and 59.

Signaling Domain

As discussed above, in certain embodiments, a CoStAR comprises a fulllength primary costimulatory receptor which can comprise anextracellular ligand binding and intracellular signaling portion of,without limitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43,CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137(41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), orEphB6. In other embodiments, the costimulatory receptor comprises achimeric protein, for instance comprising an extracellular ligandbinding domain of one of the aforementioned proteins and anintracellular signaling domain of another of the aforementionedproteins. In certain embodiments, the signaling portion of the CoStARcomprises a single signaling domain. In other embodiments, the signalingportion of the CoStAR comprises a second intracellular signaling domainsuch as but not limited to: CD2, CD27, CD28, CD40, CD134 (OX40), CD137(4-1BB), CD150 (SLAM). In certain embodiments, the first and secondintracellular signaling domains are the same. In other embodiments, thefirst and second intracellular signaling domains are different. Incertain embodiments, the costimulatory receptor is capable ofdimerization. Without being bound by theory, it is thought that CoStARsdimerize or associate with other accessory molecules for signalinitiation. In certain embodiments, CoStARs dimerize or associate withaccessory molecules through transmembrane domain interactions. Incertain embodiments, dimerization or association with accessorymolecules is assisted by costimulatory receptor interactions in theintracellular portion, and/or the extracellular portion of thecostimulatory receptor.

Transmembrane Domain

Although the main function of the transmembrane is to anchor the CoStARin the T cell membrane, in certain embodiments, the transmembrane domaininfluences CoStAR function. In certain embodiments, the transmembranedomain is comprised by the full length primary costimulatory receptordomain. In embodiments of the invention wherein the CoStAR constructcomprises an extracellular domain of one receptor and an intracellularsignaling domain of a second receptor, the transmembrane domain can bethat of the extracellular domain or the intracellular domain. In certainembodiments, the transmembrane domain is from CD4, CD8a, CD28, or ICOS.Gueden et al. associated use of the ICOS transmembrane domain withincreased CAR T cell persistence and overall anti-tumor efficacy (GuedanS. et al., Enhancing CAR T cell persistence through ICOS and 4-1BBcostimulation. JCI Insight. 2018; 3:96976). In an embodiment, thetransmembrane domain comprises a hydrophobic a helix that spans the cellmembrane.

In an embodiment, the transmembrane domain comprises amino acids of theCD28 transmembrane domain from about amino acid 153 to about amino acid179. In another embodiment, the transmembrane domain comprises aminoacids of the CD8 transmembrane domain from about amino acid 183 to aboutamino acid 203. In certain embodiments, the CoStARs of the invention mayinclude several amino acids between the transmembrane domain andsignaling domain. For example, in one construct described herein thelink from a CD8 transmembrane domain to a signaling domain comprisesseveral amino acids of the CD8 cytoplasmic domain (e.g., amino acids204-210 of CD8).

Variants

In some embodiments, amino acid sequence variants of the antibodymoieties or other moieties provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody moiety. Amino acid sequencevariants of an antibody moiety may be prepared by introducingappropriate modifications into the nucleotide sequence encoding theantibody moiety, or by peptide synthesis. Such modifications include,for example, deletions from, and/or insertions into and/or substitutionsof residues within the amino acid sequences of the antibody moiety. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., antigen-binding.

In some embodiments, antibody binding domain moieties comprising one ormore amino acid substitutions, deletions, or insertions are provided.Sites of interest for mutational changes include the antibody bindingdomain heavy and light chain variable regions (VRs) and frameworks(FRs). Amino acid substitutions may be introduced into a binding domainof interest and the products screened for a desired activity, e.g.,retained/improved antigen binding or decreased immunogenicity. Incertain embodiments, amino acid substitutions may be introduced into oneor more of the primary co-stimulatory receptor domain (extracellular orintracellular), secondary costimulatory receptor domain, orextracellular co-receptor domain. Accordingly, the invention encompassesCoStAR proteins and component parts particularly disclosed herein aswell as variants thereof, i.e. CoStAR proteins and component partshaving at least 75%, at least 80%, at least 85%, at least 87%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% sequenceidentity to the amino acid sequences particularly disclosed herein. Theterms “percent similarity,” “percent identity,” and “percent homology”when referring to a particular sequence are used as set forth in theUniversity of Wisconsin GCG software program BestFit. Other algorithmsmay be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method ofAltschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which usesthe method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448).

Particular amino acid sequence variants may differ from a referencesequence by insertion, addition, substitution or deletion of 1 aminoacid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, avariant sequence may comprise the reference sequence with 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. Forexample, 5, 10, 15, up to 20, up to 30 or up to 40 residues may beinserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a referencesequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativesubstitutions. Conservative substitutions involve the replacement of anamino acid with a different amino acid having similar properties. Forexample, an aliphatic residue may be replaced by another aliphaticresidue, a non-polar residue may be replaced by another non-polarresidue, an acidic residue may be replaced by another acidic residue, abasic residue may be replaced by another basic residue, a polar residuemay be replaced by another polar residue or an aromatic residue may bereplaced by another aromatic residue. Conservative substitutions may,for example, be between amino acids within the following groups:

Conservative substitutions are shown in the Table below.

Original Preferred Residue Exemplary Substitutions Substitutions Ala (A)Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp; Lys;Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu AsnGlu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile;Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; IleLeu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) ThrThr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; SerPhe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu,Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp,Glu; d. basic: His, Lys, Arg; e. residues that influence chainorientation: Gly, Pro; aomatic: Trp, Tyr, Phe. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class.

Cells

The cells used in the present invention may be any lymphocyte that isuseful in adoptive cell therapy, such as a T-cell or a natural killer(NK) cell, an NKT cell, a gamma/delta T-cell or T regulatory cell. Thecells may be allogeneic or autologous to the patient.

T cells or T lymphocytes are a type of lymphocyte that have a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below. Cytotoxic T cells (TCcells, or CTLs) destroy virally infected cells and tumor cells, and arealso implicated in transplant rejection. CTLs express the CD8 moleculeat their surface.

These cells recognize their targets by binding to antigen associatedwith MHC class I, which is present on the surface of all nucleatedcells. Through IL-10, adenosine and other molecules secreted byregulatory T cells, the CD8+ cells can be inactivated to an anergicstate, which prevent autoimmune diseases such as experimental autoimmuneencephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO. Regulatory T cells(Treg cells), formerly known as suppressor T cells, are crucial for themaintenance of immunological tolerance. Their major role is to shut downT cell-mediated immunity toward the end of an immune reaction and tosuppress auto-reactive T cells that escaped the process of negativeselection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells. Naturally occurring Tregcells (also known as CD4⁺CD25⁺FoxP3⁺ Treg cells) arise in the thymus andhave been linked to interactions between developing T cells with bothmyeloid (CD11c⁺) and plasmacytoid (CD123⁺) dendritic cells that havebeen activated with TSLP. Naturally occurring Treg cells can bedistinguished from other T cells by the presence of an intracellularmolecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells orTh3 cells) may originate during a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell whichform part of the innate immune system. NK cells provide rapid responsesto innate signals from virally infected cells in an MHC independentmanner. NK cells (belonging to the group of innate lymphoid cells) aredefined as large granular lymphocytes (LGL) and constitute the thirdkind of cells differentiated from the common lymphoid progenitorgenerating B and T lymphocytes.

In certain embodiments, therapeutic cells of the invention compriseautologous cells engineered to express a CoStAR. In certain embodiments,therapeutic cells of the invention comprise allogeneic cells engineeredto express a CoStAR. Autologous cells expressing CoStARs may beadvantageous in avoiding graft-versus-host disease (GVHD) due toTCR-mediated recognition of recipient alloantigens. Also, the immunesystem of a CoStAR recipient could attack the infused CoStAR cells,causing rejection. In certain embodiments, to prevent GVHD, and toreduce rejection, endogenous TcR is removed from allogeneic CoStAR cellsby genome editing.

Nucleic Acids

An aspect of the invention provides a nucleic acid sequence of theinvention, encoding any of the CoStARs, polypeptides, or proteinsdescribed herein (including functional portions and functional variantsthereof). As used herein, the terms “polynucleotide”, “nucleotide”, and“nucleic acid” are intended to be synonymous with each other. It will beunderstood by a skilled person that numerous different polynucleotidesand nucleic acids can encode the same polypeptide as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thepolynucleotides described here to reflect the codon usage of anyparticular host organism in which the polypeptides are to be expressed,e.g. codon optimisation. Nucleic acids according to the invention maycomprise DNA or RNA. They may be single stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotides maybe modified by any method available in the art. Such modifications maybe carried out in order to enhance the in vivo activity or life span ofpolynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

The nucleic acid sequence may encode the protein sequence shown as SEQID NO:2 or a variant thereof. The nucleotide sequence may comprise acodon optimised nucleic acid sequence shown engineered for expression inhuman cells.

The invention also provides a nucleic acid sequence which comprises anucleic acid sequence encoding a CoStAR and a further nucleic acidsequence encoding a T-cell receptor (TCR) and/or chimeric antigenreceptor (CAR).

The nucleic acid sequences may be joined by a sequence allowingco-expression of the two or more nucleic acid sequences. For example,the construct may comprise an internal promoter, an internal ribosomeentry sequence (IRES) sequence or a sequence encoding a cleavage site.The cleavage site may be self-cleaving, such that when the polypeptideis produced, it is immediately cleaved into the discrete proteinswithout the need for any external cleavage activity. Variousself-cleaving sites are known, including the Foot- and Mouth diseasevirus (FMDV) and the 2A self-cleaving peptide. The co-expressingsequence may be an internal ribosome entry sequence (IRES). Theco-expressing sequence may be an internal promoter.

Vectors

In an aspect, the present invention provides a vector which comprises anucleic acid sequence or nucleic acid construct of the invention.

Such a vector may be used to introduce the nucleic acid sequence(s) ornucleic acid construct(s) into a host cell so that it expresses one ormore CoStAR(s) according to the first aspect of the invention and,optionally, one or more other proteins of interest (POI), for example aTCR or a CAR. The vector may, for example, be a plasmid or a viralvector, such as a retroviral vector or a lentiviral vector, or atransposon-based vector or synthetic mRNA.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties.

Vectors derived from retroviruses, such as the lentivirus, are suitabletools to achieve long-term gene transfer since they allow long-term,stable integration of a transgene or transgenes and its propagation indaughter cells. The vector may be capable of transfecting or transducinga lymphocyte including a T cell or an NK cell. The present inventionalso provides vectors in which a nucleic acid of the present inventionis inserted. The expression of natural or synthetic nucleic acidsencoding a CoStAR, and optionally a TCR or CAR is typically achieved byoperably linking a nucleic acid encoding the CoStAR and TCR/CARpolypeptide or portions thereof to one or more promoters, andincorporating the construct into an expression vector.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1a(EF-1a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, MSCVpromoter, MND promoter, an avian leukemia virus promoter, anEpstein-Barr virus immediate early promoter, a Rous sarcoma viruspromoter, as well as human gene promoters such as, but not limited to,the actin promoter, the myosin promoter, the hemoglobin promoter, andthe creatine kinase promoter.

The vectors can be suitable for replication and integration ineukaryotic cells. Typical cloning vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals, see also, WO 01/96584; WO 01/29058; andU.S. Pat. No. 6,326,193). In some embodiments, the constructs expressedare as shown in SEQ ID NOS:32-65 and 67-79. In some embodiments thenucleic acids are multi-cistronic constructs that permit the expressionof multiple transgenes (e.g., CoStAR and a TCR and/or CAR etc.) underthe control of a single promoter. In some embodiments, the transgenes(e.g., CoStAR and a TCR and/or CAR etc.) are separated by aself-cleaving 2A peptide. Examples of 2A peptides useful in the nucleicacid constructs of the invention include F2A, P2A, T2A and E2A. In otherembodiments of the invention, the nucleic acid construct of theinvention is a multi-cistronic construct comprising two promoters; onepromoter driving the expression of CoStAR and the other promoter drivingthe expression of the TCR or CAR. In some embodiments, the dual promoterconstructs of the invention are uni-directional. In other embodiments,the dual promoter constructs of the invention are bi-directional. Inorder to assess the expression of the CoStAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or transduced through viralvectors.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g.,immune effector cells, e.g., T cells or NK cells) is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. T cell may be collected at an apheresis center and cellstorage facility where T cells can be harvested, maintained, and easilytransferred. The T cells can be cryopreserved and stored for later use.An acceptable duration of storage may be determined and validated andcan be up to 6 months, up to a year, or longer.

In another aspect, Tumor infiltrating cells (TILs) are isolated and/orexpanded from a tumor, for example by a fragmented, dissected, or enzymedigested tumor biopsy or mass. The TILs may be produced in a two-stageprocess using a tumor biopsy as the starting material: Stage 1(generally performed over 2-3 hours) initial collection and processingof tumor material using dissection, enzymatic digestion andhomogenization to produce a single cell suspension which can be directlycryopreserved to stabilize the starting material for subsequentmanufacture and Stage 2 which can occur days or years later. Stage 2 maybe performed over 4 weeks, which may be a continuous process startingwith thawing of the product of Stage 1 and growth of the TIL out of thetumor starting material (about 2 weeks) followed by a rapid expansionprocess of the TIL cells (about 2 weeks) to increase the amount of cellsand therefore dose. The TILs maybe concentrated and washed prior toformulation as a liquid suspension of cells.

The TIL population can be transduced at any point following collection.In certain embodiments, a cryopreserved TIL population is transduced toexpress a CoStAR following thawing. In certain embodiments, a TILpopulation is transduced to express a CoStAR during outgrowth or initialexpansion from tumor starting material. In certain embodiments, a TILpopulation is transduced to express a CoStAR during REP, for example butnot limited to from about day 8 to about day 10 of REP. An exemplary TILpreparation is described in Applicant's U.S. patent application Ser. No.62/951,559, filed Dec. 20, 2019.

A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+,CD45RA+, and CD45RO+T cells, can be further isolated by positive ornegative selection techniques. For example, in one aspect, T cells areisolated by incubation with anti-CD3/anti-CD28-conjugated beads, such asDYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positiveselection of the desired T cells. In one aspect, the time period isabout 30 minutes. In a further aspect, the time period ranges from 30minutes to 36 hours or longer and all integer values there between. In afurther aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours.In yet another preferred aspect, the time period is 10 to 24 hours. Inone aspect, the incubation time period is 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD16, HLA-DR, andCD8. In certain aspects, it may be desirable to enrich for or positivelyselect for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+.Alternatively, in certain aspects, T regulatory cells are depleted byanti-CD25 conjugated beads or other similar method of selection.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

A specific subpopulation of CoStAR effector cells that specifically bindto a target antigen can be enriched for by positive selectiontechniques. For example, in some embodiments, effector cells areenriched for by incubation with target antigen-conjugated beads for atime period sufficient for positive selection of the desired abTCReffector cells. In some embodiments, the time period is about 30minutes. In some embodiments, the time period ranges from 30 minutes to36 hours or longer (including all ranges between these values). In someembodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. Insome embodiments, the time period is 10 to 24 hours. In someembodiments, the incubation time period is 24 hours. For isolation ofeffector cells present at low levels in the heterogeneous cellpopulation, use of longer incubation times, such as 24 hours, canincrease cell yield. Longer incubation times may be used to isolateeffector cells in any situation where there are few effector cells ascompared to other cell types. The skilled artisan would recognize thatmultiple rounds of selection can also be used in the context of thisinvention.

T cells for stimulation can also be frozen after a washing step. Afterthe washing step that removes plasma and platelets, the cells may besuspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serumalbumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20%Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human SerumAlbumin, and 7.5% DMSO or other suitable cell freezing media containingfor example, Hespan and PlasmaLyte A, the cells then are frozen to −80°C. at a rate of 1° per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

Allogenic CoStAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of endogenous T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional endogenous TCR can be, e.g., engineeredsuch that it does not express any functional TCR on its surface,engineered such that it does not express one or more subunits thatcomprise a functional TCR (e.g., engineered such that it does notexpress (or exhibits reduced expression) of TCR alpha, TCR beta, TCRgamma, TCR delta, TCR epsilon, and/or TCR zeta) or engineered such thatit produces very little functional TCR on its surface. Alternatively,the T cell can express a substantially impaired TCR, e.g., by expressionof mutated or truncated forms of one or more of the subunits of the TCR.The term “substantially impaired TCR” means that this TCR will notelicit an adverse immune reaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some aspects,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II. Modified T cellsthat lack expression of a functional TCR and/or HLA can be obtained byany suitable means, including a knock out or knock down of one or moresubunit of TCR or HLA. For example, the T cell can include a knock downof TCR and/or HLA using siRNA, shRNA, clustered regularly interspacedshort palindromic repeats (CRISPR) transcription-activator like effectornuclease (TALEN), or zinc finger endonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. a cellengineered by any method described herein. For example, the cell can bea cell that does not express or expresses at low levels an inhibitorymolecule, e.g., that can decrease the ability of a CoStAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, Ga19, adenosine, and TGFRbeta. Inhibition of an inhibitory molecule, e.g., by inhibition at theDNA, RNA or protein level, can optimize a CAR-expressing cellperformance. In embodiments, an inhibitory nucleic acid, e.g., aninhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, aclustered regularly interspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used.

Use of siRNA or shRNA to inhibit endogenous TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, Ga19, adenosine, and TGFR beta), in a T cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem. Exemplary shRNAs that downregulate expression of components ofthe TCR are described, e.g., in US Publication No.: 2012/0321667.Exemplary siRNA and shRNA that downregulate expression of HLA class Iand/or HLA class II genes are described, e.g., in U.S. publication No.:US 2007/0036773.

CRISPR to Inhibit TCR or HLA

“CRISPR” or “CRISPR to inhibit TCR and/or HLA” as used herein refers toa set of clustered regularly interspaced short palindromic repeats, or asystem comprising such a set of repeats. “Cas”, as used herein, refersto a CRISPR-associated protein. A “CRISPR/Cas” system refers to a systemderived from CRISPR and Cas which can be used to silence or mutate a TCRand/or HLA gene, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GALS, adenosine, and TGFR beta).

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

Activation and Expansion of T Cells

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody can be used. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

In some embodiments, expansion can be performed using flasks orcontainers, or gas-permeable containers known by those of skill in theart and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, or 14 days, about 7 days to about 14 days, about 8 daysto about 14 days, about 9 days to about 14 days, about 10 days to about14 days, about 11 days to about 14 days, about 12 days to about 14 days,or about 13 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 14 days.

In certain embodiments, the expansion can be performed usingnon-specific T-cell receptor stimulation in the presence ofinterleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T-cellreceptor stimulus can include, for example, an anti-CD3 antibody, suchas about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(commercially available from Ortho-McNeil, Raritan, N.J. or MiltenyiBiotech, Auburn, Calif.) or UHCT-1 (commercially available fromBioLegend, San Diego, Calif., USA). CoStAR cells can be expanded invitro by including one or more antigens, including antigenic portionsthereof, such as epitope(s), of a cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3.mu·M MART-1:26-35 (27L) or gp100:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. CoStAR cells may also be rapidlyexpanded by restimulation with the same antigen(s) of the cancer pulsedonto HLA-A2-expressing antigen-presenting cells. Alternatively, theCoStAR cells can be further stimulated with, e.g., example, irradiated,autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytesand IL-2. In some embodiments, the stimulation occurs as part of theexpansion. In some embodiments, the expansion occurs in the presence ofirradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneiclymphocytes and IL-2.

In certain embodiments, the cell culture medium comprises IL-2. In someembodiments, the cell culture medium comprises about 1000 IU/mL, about1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In certain embodiments, the cell culture medium comprises OKT3 antibody.In some embodiments, the cell culture medium comprises about 0.1 ng/mL,about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL,about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL or between0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL,between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between50 ng/mL and 100 ng/mL of OKT3 antibody.

In certain embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the expansion. In some embodiments,IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof canbe included during the expansion. In some embodiments, a combination ofIL-2, IL-15, and IL-21 are employed as a combination during theexpansion. In some embodiments, IL-2, IL-15, and IL-21 as well as anycombinations thereof can be included.

In certain embodiments, the expansion can be conducted in a supplementedcell culture medium comprising IL-2, OKT-3, and antigen-presentingfeeder cells.

In certain embodiments, the expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15,or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 300IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15,or about 180 IU/mL of IL-15.

In some embodiments, the expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mLof IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, orabout 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mLof IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL of IL-21 to about1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In some embodiments, thecell culture medium comprises about 1 IU/mL of IL-21, or about 0.5 IU/mLof IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of CoStAR cells to PBMCs and/orantigen-presenting cells in the expansion is about 1 to 25, about 1 to50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175,about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400,or about 1 to 500, or between 1 to 50 and 1 to 300, or between 1 to 100and 1 to 200.

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle:cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

Preparation of CoStAR Cells

Viral- and non-viral-based genetic engineering tools can be used togenerate CoStAR cells, including without limitation T cells, NK cellsresulting in permanent or transient expression of therapeutic genes.Retrovirus-based gene delivery is a mature, well-characterizedtechnology, which has been used to permanently integrate CARs into thehost cell genome (Scholler J., e.g. Decade-long safety and function ofretroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med.2012; 4:132ra53; Rosenberg S. A. et al., Gene transfer intohumans—immunotherapy of patients with advanced melanoma, usingtumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323:570-578)

Non-viral DNA transfection methods can also be used. For example, Singhet al describes use of the Sleeping Beauty (SB) transposon systemdeveloped to engineer CAR T cells (Singh H., et al., Redirectingspecificity of T-cell populations for CD19 using the Sleeping Beautysystem. Cancer Res. 2008; 68:2961-2971) and is being used in clinicaltrials (see e.g., ClinicalTrials.gov: NCT00968760 and NCT01653717). Thesame technology is applicable to engineer CoStARs cells.

Multiple SB enzymes have been used to deliver transgenes. Matesdescribes a hyperactive transposase (SB100X) with approximately 100-foldenhancement in efficiency when compared to the first-generationtransposase. SB100X supported 35-50% stable gene transfer in humanCD34(+) cells enriched in hematopoietic stem or progenitor cells. (MatesL. et al., Molecular evolution of a novel hyperactive Sleeping Beautytransposase enables robust stable gene transfer in vertebrates. Nat.Genet. 2009; 41:753-761) and multiple transgenes can be delivered frommulticistronic single plasmids (e.g., Thokala R. et al., Redirectingspecificity of T cells using the Sleeping Beauty system to expresschimeric antigen receptors by mix-and-matching of VL and VH domainstargeting CD123+ tumors. PLoS ONE. 2016; 11:e0159477) or multipleplasmids (e.g., Hurton L. V. et al., Tethered IL-15 augments antitumoractivity and promotes a stem-cell memory subset in tumor-specific Tcells. Proc. Natl. Acad. Sci. USA. 2016; 113:E7788-E7797). Such systemsare used with CoStARs of the invention.

Morita et al, describes the piggyBac transposon system to integratelarger transgenes (Morita D. et al., Enhanced expression of anti-CD19chimeric antigen receptor in piggyBac transposon-engineered T cells.Mol. Ther. Methods Clin. Dev. 2017; 8:131-140) Nakazawa et al. describesuse of the system to generate EBV-specific cytotoxic T-cells expressingHER2-specific chimeric antigen receptor (Nakazawa Y et al,PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxicT-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther.2011; 19:2133-2143). Manuri et al used the system to generate CD-19specific T cells (Manuri P. V. R. et al., piggyBactransposon/transposase system to generate CD19-specific T cells for thetreatment of B-lineage malignancies. Hum. Gene Ther. 2010; 21:427-437).

Transposon technology is easy and economical. One potential drawback isthe longer expansion protocols currently employed may result in T celldifferentiation, impaired activity and poor persistence of the infusedcells. Monjezi et al describe development minicircle vectors thatminimize these difficulties through higher efficiency integrations(Monjezi R. et al., Enhanced CAR T-cell engineering using non-viralSleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186-194). These transposon technologies can be used for CoStARs ofthe invention.

Pharmaceutical Compositions

The present invention also relates to a pharmaceutical compositioncontaining a vector or a CoStAR expressing cell of the inventiontogether with a pharmaceutically acceptable carrier, diluent orexcipient, and optionally one or more further pharmaceutically activepolypeptides and/or compounds.

In some embodiments, a pharmaceutical composition is provided comprisinga CoStAR described above and a pharmaceutically acceptable carrier. Insome embodiments, a pharmaceutical composition is provided comprising anucleic acid encoding a CoStAR according to any of the embodimentsdescribed above and a pharmaceutically acceptable carrier. In someembodiments, a pharmaceutical composition is provided comprising aneffector cell expressing a CoStAR described above and a pharmaceuticallyacceptable carrier. Such a formulation may, for example, be in a formsuitable for intravenous infusion.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

An aspect of the invention provides a population of modified T cellsexpressing a recombinant CoStAR. A suitable population may be producedby a method described above.

The population of modified T cells may be for use as a medicament. Forexample, a population of modified T cells as described herein may beused in cancer immunotherapy therapy, for example adoptive T celltherapy.

Other aspects of the invention provide the use of a population ofmodified T cells as described herein for the manufacture of a medicamentfor the treatment of cancer, a population of modified T cells asdescribed herein for the treatment of cancer, and a method of treatmentof cancer may comprise administering a population of modified T cells asdescribed herein to an individual in need thereof.

The population of modified T cells may be autologous i.e. the modified Tcells were originally obtained from the same individual to whom they aresubsequently administered (i.e. the donor and recipient individual arethe same). A suitable population of modified T cells for administrationto the individual may be produced by a method comprising providing aninitial population of T cells obtained from the individual, modifyingthe T cells to express a cAMP PDE or fragment thereof and an antigenreceptor which binds specifically to cancer cells in the individual, andculturing the modified T cells.

The population of modified T cells may be allogeneic i.e. the modified Tcells were originally obtained from a different individual to theindividual to whom they are subsequently administered (i.e. the donorand recipient individual are different). The donor and recipientindividuals may be HLA matched to avoid GVHD and other undesirableimmune effects. A suitable population of modified T cells foradministration to a recipient individual may be produced by a methodcomprising providing an initial population of T cells obtained from adonor individual, modifying the T cells to express a CoStAR which bindsspecifically to cancer cells in the recipient individual, and culturingthe modified T cells.

Following administration of the modified T cells, the recipientindividual may exhibit a T cell mediated immune response against cancercells in the recipient individual. This may have a beneficial effect onthe cancer condition in the individual.

Cancer conditions may be characterised by the abnormal proliferation ofmalignant cancer cells and may include leukaemias, such as AML, CML, ALLand CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma andmultiple myeloma, and solid cancers such as sarcomas, skin cancer,melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer,ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervicalcancer, liver cancer, head and neck cancer, oesophageal cancer, pancreascancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer,cancer of the gall bladder and biliary tracts, thyroid cancer, thymuscancer, cancer of bone, and cerebral cancer, as well as cancer ofunknown primary (CUP).

Cancer cells within an individual may be immunologically distinct fromnormal somatic cells in the individual (i.e. the cancerous tumor may beimmunogenic). For example, the cancer cells may be capable of elicitinga systemic immune response in the individual against one or moreantigens expressed by the cancer cells. The tumor antigens that elicitthe immune response may be specific to cancer cells or may be shared byone or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal,such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

In preferred embodiments, the individual is a human. In other preferredembodiments, non-human mammals, especially mammals that areconventionally used as models for demonstrating therapeutic efficacy inhumans (e.g. murine, primate, porcine, canine, or rabbit animals) may beemployed.

Method of Treatment

The term “therapeutically effective amount” refers to an amount of aCoStAR or composition comprising a CoStAR as disclosed herein, effectiveto “treat” a disease or disorder in an individual. In the case ofcancer, the therapeutically effective amount of a CoStAR or compositioncomprising a CoStAR as disclosed herein can reduce the number of cancercells; reduce the tumor size or weight; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent a CoStAR or composition comprising a CoStAR as disclosedherein can prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. In some embodiments, the therapeuticallyeffective amount is a growth inhibitory amount. In some embodiments, thetherapeutically effective amount is an amount that improves progressionfree survival of a patient. In the case of infectious disease, such asviral infection, the therapeutically effective amount of a CoStAR orcomposition comprising a CoStAR as disclosed herein can reduce thenumber of cells infected by the pathogen; reduce the production orrelease of pathogen-derived antigens; inhibit (i.e., slow to some extentand preferably stop) spread of the pathogen to uninfected cells; and/orrelieve to some extent one or more symptoms associated with theinfection. In some embodiments, the therapeutically effective amount isan amount that extends the survival of a patient.

Cells, including T and NK cells, expressing CoStARs for use in themethods of the present may either be created ex vivo either from apatient's own peripheral blood (autologous), or in the setting of ahaematopoietic stem cell transplant from donor peripheral blood(allogenic), or peripheral blood from an unconnected donor (allogenic).Alternatively, T-cells or NK cells may be derived from ex-vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to T-cells or NK cells. In these instances, T-cells expressing aCoStAR and, optionally, a CAR and/or TCR, are generated by introducingDNA or RNA coding for the CoStAR and, optionally, a CAR and/or TCR, byone of many means including transduction with a viral vector,transfection with DNA or RNA.

T or NK cells expressing a CoStAR of the present invention and,optionally, expressing a TCR and/or CAR may be used for the treatment ofhaematological cancers or solid tumors.

A method for the treatment of disease relates to the therapeutic use ofa vector or cell, including a T or NK cell, of the invention. In thisrespect, the vector, or T or NK cell may be administered to a subjecthaving an existing disease or condition in order to lessen, reduce orimprove at least one symptom associated with the disease and/or to slowdown, reduce or block the progression of the disease. The method of theinvention may cause or promote T-cell mediated killing of cancer cells.The vector, or T or NK cell according to the present invention may beadministered to a patient with one or more additional therapeuticagents. The one or more additional therapeutic agents can beco-administered to the patient. By “co-administering” is meantadministering one or more additional therapeutic agents and the vector,or T or NK cell of the present invention sufficiently close in time suchthat the vector, or T or NK cell can enhance the effect of one or moreadditional therapeutic agents, or vice versa. In this regard, thevectors or cells can be administered first and the one or moreadditional therapeutic agents can be administered second, or vice versa.Alternatively, the vectors or cells and the one or more additionaltherapeutic agents can be administered simultaneously. Oneco-administered therapeutic agent that may be useful is IL-2, as this iscurrently used in existing cell therapies to boost the activity ofadministered cells. However, IL-2 treatment is associated with toxicityand tolerability issues.

As mentioned, for administration to a patient, the CoStAR effector cellscan be allogeneic or autologous to the patient. In certain embodiments,allogeneic cells are further genetically modified, for example by geneediting, so as to minimize or prevent GVHD and/or a patient's immuneresponse against the CoStAR cells.

The CoStAR effector cells are used to treat cancers and neoplasticdiseases associated with a target antigen. Cancers and neoplasticdiseases that may be treated using any of the methods described hereininclude tumors that are not vascularized, or not yet substantiallyvascularized, as well as vascularized tumors. The cancers may comprisenon-solid tumors (such as hematological tumors, for example, leukemiasand lymphomas) or may comprise solid tumors. Types of cancers to betreated with the CoStAR effector cells of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, plasmacytoma, Waldenstrom'smacroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairycell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include adrenocorticalcarcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer,breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullarythyroid carcinoma and papillary thyroid carcinoma), pheochromocytomassebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer (e.g., cervical carcinoma and pre-invasive cervicaldysplasia), colorectal cancer, cancer of the anus, anal canal, oranorectum, vaginal cancer, cancer of the vulva (e.g., squamous cellcarcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma),penile cancer, oropharyngeal cancer, esophageal cancer, head cancers(e.g., squamous cell carcinoma), neck cancers (e.g., squamous cellcarcinoma), testicular cancer (e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor,fibroma, fibroadenoma, adenomatoid tumors, and lipoma), bladdercarcinoma, kidney cancer, melanoma, cancer of the uterus (e.g.,endometrial carcinoma), urothelial cancers (e.g., squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer,and urinary bladder cancer), and CNS tumors (such as a glioma (such asbrainstem glioma and mixed gliomas), glioblastoma (also known asglioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T cell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988).

Combination Therapies

A CoStAR-expressing cell described herein may be used in combinationwith other known agents and therapies. Administered “in combination”, asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CoStAR-expressing cell described herein and the at least oneadditional therapeutic agent can be administered simultaneously, in thesame or in separate compositions, or sequentially. For sequentialadministration, the CAR-expressing cell described herein can beadministered first, and the additional agent can be administered second,or the order of administration can be reversed.

The CoStAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CoStAR therapycan be administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the therapy and the additional agent(e.g., second or third agent), or all, can be administered in an amountor dose that is higher, lower or the same than the amount or dosage ofeach agent used individually, e.g., as a monotherapy. In certainembodiments, the administered amount or dosage of the CoStAR therapy,the additional agent (e.g., second or third agent), or all, is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50%) thanthe amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CoStARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CoStAR-expressing cell described herein may beused in a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation,peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971.

In certain instances, compounds of the present invention are combinedwith other therapeutic agents, such as other anti-cancer agents,anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

In one embodiment, a CoStAR-expressing cell described herein can be usedin combination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab,tositumomab, brentuximab), an antimetabolite (including, e.g., folicacid antagonists, pyrimidine analogs, purine analogs and adenosinedeaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include busulfan (Myleran®), busulfan injection (Busulfex®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), Idarubicin(Idamycin®), mitoxantrone (Novantrone®), Gemtuzumab Ozogamicin(Mylotarg®).

In embodiments, general chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Treatments can be evaluated, for example, by tumor regression, tumorweight or size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,quality of life, protein expression and/or activity. Approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of response through radiological imaging.

Sequences

The following sequences include complete CoStARs and CoStAR componentsand are non-limiting. Components include signal peptides (SP), bindingdomains (BD), linkers, spacers and transmembrane domains (STM), a CD28transmembrane fragment without extracellular or intracellular sequences(STM-CD28TM), intracellular signal domains (SD) and CD40 domains andmotifs. SEQ ID NOS:33-108 comprise CoStARs with N-terminal signalpeptides. Component locations within whole proteins can be confirmedfrom GenBank and other sources. The constructs and components areillustrative as to precise sizes and extents and components can be frommore than one source. Where there is more than one intracellularsignaling domain or signaling fragment, the multiple domains can be inany order. It will be understood that whereas certain proteins maycomprise N-terminal signal peptides when expressed, those signalpeptides are cleaved and may be imprecisely cleaved when the proteinsare expressed, and that the resulting proteins from which signalpeptides are removed comprise binding domains having variation of up toabout five amino acids in the location of the N-terminal amino acid.

TABLE of Sequences ID NO: Components Sequence 1 SP-OSMMGVLLTQRTL LSLVLALLFP SMASM 2 SP-PD1 MQIPQAPWPV VWAVLQLGWR PGW 3 SP-TGITMRWCLLLIWA QGLRQAPLAS G 4 BD1-MOV19QVQLQQSGAE LVKPGASVKI SCKASGYSFT GYFMNWVKQS HGKSLEWIGRIHPYDGDTFY NQNFKDKATL TVDKSSNTAH MELLSLTSED FAVYYCTRYDGSRAMDYWGQ GTTVTVSSGG GGSGGGGSGG GGSDIELTQS PASLAVSLGQRAIISCKASQ SVSFAGTSLM HWYHQKPGQQ PKLLIYRASN LEAGVPTRFSGSGSKTDFTL NIHPVEEEDA ATYYCQQSRE YPYTFGGGTK LEIK 5 BD1-MFE23QVQLQQSGAE LVRSGTSVKL SCTASGFNIK DSYMHWLRQG PEQGLEWIGWIDPENGDTEY APKFQGKATF TTDTSSNTAY LQLSSLTSED TAVYYCNEGTPTGPYYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSENVLT QSPAIMSASPGEKVTITCSA SSSVSYMHWF QQKPGTSPKL WIYSTSNLAS GVPARFSGSGSGTSYSLTIS RMEAEDAATY YCQQRSSYPL TFGAGTKLEL KR 6 BD1-PD1RPGWFLDSPD RPWNPPTFSP ALLVVTEGDN ATFTCSFSNT SESFVLNWYRMSPSNQTDKL AAFPEDRSQP GQDCRFRVTQ LPNGRDFHMS VVRARRNDSGTYLCGAISLA PKAQIKESLR AELRVTERRA EVPTAH 7 BD1-TIGITMMTGTIETTG NISAEKGGSIILQCHLSSTT AQVTQVNWEQ QDQLLAICNADLGWHISPSF KDRVAPGPGL GLTLQSLTVN DTGEYFCIYH TYPDGTYTGRIFLEVLESSV AEHGARFQIP 8 3xA3xGS AAAGSGGSG 9 BD2-PD1RPGWFLDSPD RPWNPPTFSP ALLVVTEGDN ATFTCSFSNT SESFVLNWYRMSPSNQTDKL AAFPEDRSQP GQDCRFRVTQ LPNGRDFHMS VVRARRNDSGTYLCGAISLA PKAQIKESLR AELRVTERRA EVPTAH 10 BD2-TGITMMTGTIETTG NISAEKGGSI ILQCHLSSTT AQVTQVNWEQ QDQLLAICNADLGWHISPSF KDRVAPGPGL GLTLQSLTVN DTGEYFCIYH TYPDGTYTGRIFLEVLESSV AEHGARFQIP 11 STM-spCD28ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYGNYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWV 12STM-spCD8 FVPVFLPAKP TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGLDFACDIYIWA PLAGTCGVLL LSLVITLYCN HRN 13 STM-spIG4ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQEDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGKM FWVLVVVGGV LACYSLLVTV AFIIFWV 14STM-sCD28TM CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWV 15 STM-CD28TMFWVLVVVGGV LACYSLLVTV AFIIFWV 16 Sig-CD28RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 17 Sig-CD137RFSVVKRGRK KLLYIFKQPF MRPVQTTQEE DGCSCRFPEE EEGGCE 18 Sig-CD134RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 19 Sig-CD2KRKKQRSRRN DEELETRAHR VATEERGRKP HQIPASTPQN PATSQHPPPPPGHRSQAPSH RPPPPGHRVQ HQPQKRPPAP SGTQVHQQKG PPLPRPRVQP KPPHGAAENS LSPSSN20 Sig GITR QLGLHIWQLR SQCMWPRETQ LLLEVPPSTE DARSCQFPEEERGERSAEEK GRLGDLWV 21 Sig-CD29KLLMIIHDRR EFAKFEKEKM NAKWDTGENP IYKSAVTTVV NPKYEGK 22 Sig-CD150RRRGKTNHYQ TTVEKKSLTI YAQVQKPGPL QKKLDSFPAQ DPCTTIYVAATEPVPESVQE TNSITVYASV TLPES 23 CD40KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 24CD40_tandem KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 25 CD40_P227AKKVAKKPTNK AAHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ 26SH3_motif KPTNKAPH 27 TRAF2_motif1 PKQE 28 TRAF2_motif2 PVQE 29TRAF2_motif3 SVQE 30 TRAF6_motif QEPQEINFP 31 PKA_motif1 KKPTNKA 32PKA_motif2 SRISVQE 33 CTP194 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSGOSM_MFE23_spCD28 TSVKLSCTAS _CD28_CD40GFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTSSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 34 OSM_MFE23MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG _spCD28_CD40 TSVKLSCTASGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTSSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 35 OSM_MFE23_spCD28MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG _CD137_CD40 TSVKLSCTASGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ GKATFTTDTSSNTAYLQLSS LTSEDTAVYY CNEGTPTGPY YFDYWGQGTT VTVSSGGGGSGGGGSGGGGS ENVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPGTSPKLWIYST SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQRSSYPLTFGAG TKLELKRAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSYNLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNESVTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSPLFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRFSVV KRGRKKLLYIFKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 36 OSM_MFE23_spCD28MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG _CD134_CD40 TSVKLSCTASGFNIKDSYMH WLRQGPEQGL EWIGWIDPEN GDTEYAPKFQ 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OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD28_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 65OSM_MO V19_spCD28 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG_CD137_CD40 ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFKDKATLTVDKS SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRFS VVKRGRKKLLYIFKQPFMRP VQTTQEEDGC SCRFPEEEEG GCEKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 66 OSM_MO V19_spCD28MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD134_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRRD QRLPPDAHKPPGGGSFRTPI QEEQADAHST LAKIKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 67 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD28_CD2_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKRK KQRSRRNDEELETRAHRVAT EERGRKPHQI PASTPQNPAT SQHPPPPPGH RSQAPSHRPPPPGHRVQHQP QKRPPAPSGT QVHQQKGPPL PRPRVQPKPP HGAAENSLSPSSNKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ68 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD28ASVKISCKAS _GITR_CD40 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFKDKATLTVDKS SNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVQLG LHIWQLRSQCMWPRETQLLL EVPPSTEDAR SCQFPEEERG ERSAEEKGRL GDLWVKKVAKKPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 69OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD28 ASVKISCKAS_CD29_CD40 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVKLL MIIHDRREFAKFEKEKMNAK WDTGENPIYK SAVTTVVNPK YEGKKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 70 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD28 ASVKISCKAS_CD150_CD40 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKYSYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGNESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCPSPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRRR GKTNHYQTTVEKKSLTIYAQ VQKPGPLQKK LDSFPAQDPC TTIYVAATEP VPESVQETNSITVYASVTLP ESKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ 71 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_CD28_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ72 OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_CD40ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQETLHGCQPVTQE DGKESRISVQ ERQ 73 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_CD137_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRFSVVKRGR KKLLYIFKQP FMRPVQTTQE EDGCSCRFPEEEEGGCEKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ 74 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_CD134_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRRDQRLPPD AHKPPGGGSF RTPIQEEQAD AHSTLAKIKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK SRISVQERQ 75OSM_MOV19 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_CD2_CD40ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKRKKQRSRR NDEELETRAH RVATEERGRK PHQIPASTPQNPATSQHPPP PPGHRSQAPS HRPPPPGHRV QHQPQKRPPA PSGTQVHQQKGPPLPRPRVQ PKPPHGAAEN SLSPSSNKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ 76 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spCD8_GITR_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NQLGLHIWQL RSQCMWPRET QLLLEVPPST EDARSCQFPEEERGERSAEE KGRLGDLWVK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q 77 OSM_MOV19_spCD8MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD29_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NKLLMIIHDR REFAKFEKEK MNAKWDTGEN PIYKSAVTTVVNPKYEGKKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ 78 OSM_MOV19_spCD8MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD150_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGFV PVFLPAKPTT TPAPRPPTPAPTIASQPLSL RPEACRPAAG GAVHTRGLDF ACDIYIWAPL AGTCGVLLLSLVITLYCNHR NRRRGKTNHY QTTVEKKSLT IYAQVQKPGP LQKKLDSFPAQDPCTTIYVA ATEPVPESVQ ETNSITVYAS VTLPESKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 79 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD28_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ 80 OSM_MOV19MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _spIG4_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 81 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD137_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 82 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD134_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVRRDQR LPPDAHKPPGGGSFRTPIQE EQADAHSTLA KIKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 83 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD2_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKRKKQ RSRRNDEELETRAHRVATEE RGRKPHQIPA STPQNPATSQ HPPPPPGHRS QAPSHRPPPPGHRVQHQPQK RPPAPSGTQV HQQKGPPLPR PRVQPKPPHG AAENSLSPSSNKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ 84OSM_MOV19_spIG4 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _GITR_CD40ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVQLGLH IWQLRSQCMWPRETQLLLEV PPSTEDARSC QFPEEERGER SAEEKGRLGD LWVKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 85OSM_MOV19_spIG4 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD29_CD40ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVKLLMI IHDRREFAKFEKEKMNAKWD TGENPIYKSA VTTVVNPKYE GKKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ 86 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD150_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVRRRGK TNHYQTTVEKKSLTIYAQVQ KPGPLQKKLD SFPAQDPCTT IYVAATEPVP ESVQETNSITVYASVTLPES KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETLHGCQPVTQED GKESRISVQE RQ 87 OSM_MOV19_spIG4MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD40_tandem ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAF IIFWVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQKKVA 88OSM_MOV19_spIG4 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _CD40_P227AASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKSLSLSLGKMFW VLVVVGGVLA CYSLLVTVAFIIFWVKKVAK KPTNKAAHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 89 CTP188MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA PD1_PD1_sCD28TM LLVVTEGDNA_CD28_CD40 TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG (dimeric)QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAEVPTAHCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 90 PD1_PD1_sCD28TMMQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA _CD40 LLVVTEGDNATFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQLPNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAEVPTAHCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 91TIGIT_TIGIT MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS _CD28TMIILQCHLSST _CD28_CD40 TAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPGLGLTLQSLTV NDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PFWVLVVVGGVLACYSLLVT VAFIIFWVRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q 92 TIGIT_TIGITMRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS _CD28TM_CD40 IILQCHLSSTTAQVTQVNWE QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTVNDTGEYFCIY HTYPDGTYTG RIFLEVLESS VAEHGARFQI PFWVLVVVGGVLACYSLLVT VAFIIFWVKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 93 OSM_MOV19_PD1MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _sCD28TM_CD28 ASVKISCKAS_CD40 GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGRP GWFLDSPDRP WNPPTFSPALLVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDCRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAELRVTERRAEV PTAHCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFIIFWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKKPTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ 94OSM_MOV19_PD1 MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _sCD28TM_CD40ASVKISCKAS GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGRP GWFLDSPDRP WNPPTFSPALLVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQDCRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAELRVTERRAEV PTAHCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFIIFWVKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ95 0SM_M0V19_TIGIT MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG_sCD28TM_CD28 ASVKISCKAS _CD40GYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGMM TGTIETTGNI SAEKGGSIILQCHLSSTTAQ VTQVNWEQQD QLLAICNADL GWHISPSFKD RVAPGPGLGLTLQSLTVNDT GEYFCIYHTY PDGTYTGRIF LEVLESSVAE HGARFQIPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ 96 0SM_M0V19_TIGITMGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVKPG _sCD28TM_CD40 ASVKISCKASGYSFTGYFMN WVKQSHGKSL EWIGRIHPYD GDTFYNQNFK DKATLTVDKSSNTAHMELLS LTSEDFAVYY CTRYDGSRAM DYWGQGTTVT VSSGGGGSGGGGSGGGGSDI ELTQSPASLA VSLGQRAIIS CKASQSVSFA GTSLMHWYHQKPGQQPKLLI YRASNLEAGV PTRFSGSGSK TDFTLNIHPV EEEDAATYYCQQSREYPYTF GGGTKLEIKA AAGSGGSGMM TGTIETTGNI SAEKGGSIILQCHLSSTTAQ VTQVNWEQQD QLLAICNADL GWHISPSFKD RVAPGPGLGLTLQSLTVNDT GEYFCIYHTY PDGTYTGRIF LEVLESSVAE HGARFQIPFWVLVVVGGVLA CYSLLVTVAF IIFWVKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 97 linker GGGGSGGGGS GGGGS 98Truncated NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG CytoplasmicLDSAVEVCVV domain CD28 YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTDVariant IYFCKIEVMY PPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVVVGGVLACYSL LVTVAFIIFW VRSKR 99 CD28.CD 137NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRFSVVKRG RKKLLYIFKQ PFMRPVQTTQ EEDGCSCRFP EEEEGGCE 100 CD28.CD134NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRRDQRLPP DAHKPPGGGS FRTPIQEEQA DAHSTLAKI 101 CD28.CD2NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKRKKQRSR RNDEELETRA HRVATEERGR KPHQIPASTP QNPATSQHPPPPPGHRSQAP SHRPPPPGHR VQHQPQKRPP APSGTQVHQQ KGPPLPRPRVQPKPPHGAAE NSLSPSSN 102 CD28.CD29NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKLLMIIHD RREFAKFEKE KMNAKWDTGE NPIYKSAVTT VVNPKYEGK 103 CD28.GITRNKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSQLGLHIWQ LRSQCMWPRE TQLLLEVPPS TEDARSCQFP EEERGERSAE EKGRLGDLWV 104CD28.IL2Rγ NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSERTMPRIP TLKNLEDLVT EYHGNFSAWS GVSKGLAESL QPDYSERLCLVSEIPPKGGA LGEGPGASPC NQHSPYWAPP CYTLKPET 105 CD28.CD40NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ106 CD28.CD 150 NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusionLDSAVEVCVV YGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSRRRGKTNH YQTTVEKKSL TIYAQVQKPG PLQKKLDSFP AQDPCTTIYVAATEPVPESV QETNSITVYA SVTLPES 107 CD28.CD2.CD40NKILVKQSPM LVAYDNAVNL SCKYSYNLFS REFRASLHKG fusion LDSAVEVCVVYGNYSQQLQV YSKTGFNCDG KLGNESVTFY LQNLYVNQTD IYFCKIEVMYPPPYLDNEKS NGTIIHVKGK HLCPSPLFPG PSKPFWVLVV VGGVLACYSLLVTVAFIIFW VRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAYRSKRKKQRSR RNDEELETRA HRVATEERGR KPHQIPASTP QNPATSQHPPPPPGHRSQAP SHRPPPPGHR VQHQPQKRPP APSGTQVHQQ KGPPLPRPRVQPKPPHGAAE NSLSPSSNKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ 108 CD28(IEV)IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF Variant WVLVVVGGVLACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRS 109CTP189 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA PD1_PD1_sCD28TMLLVVTEGDNA _CD28_CD40 TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG(monomeric) QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRAELRVTERRAE VPTAHPSPSP RPAGQFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ 110 CTP195MGVLLTQRTL LSLVLALLFP SMASMQVQLQ QSGAELVRSG 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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1—Production of T-Cells Expressing CoStAR

Materials and Methods

Construct design—The MFE23 CoStAR consists of an MFE23 derived singlechain antibody fragment nucleotide sequence with an oncostatin M1 leadersequence fused to the entire human CD28 nucleic acid sequence. TheCoStAR nucleotide sequence was codon optimised and gene synthesised byGenewiz Inc. The constructs were cloned into pSF.Lenti (Oxford Genetics)via an XbaI and NheI site.

Lentiviral Production—Lentiviral production was performed using athree-plasmid packaging system (Cell Biolabs, San Diego, USA) by mixing10 μg of each plasmid, plus 10 μg of the pSF.Lenti lentiviral plasmidcontaining the transgene, together in serum free RPMI containing 50 mMCaCl₂). The mixture was added dropwise to a 50% confluent monolayer of293T cells in 75 cm² flasks. The viral supernatants were collected at 48and 72 h post transfection, pooled and concentrated using LentiPaclentiviral supernatant concentration (GeneCopoeia, Rockville, Md., USA)solution according to the manufacturer's instructions. Lentiviralsupernatants were concentrated 10-fold and used to directly infectprimary human T-cells in the presence of 4 μg/ml polybrene(Sigma-Aldrich, Dorset, UK). Peripheral blood mononuclear cells wereisolated from normal healthy donors before activation for 24 hours withT-cell activation and expansion beads (Invitrogen) according to themanufacturer's instructions before addition of lentiviral supernatants.

Cell transduction was assessed 96 hours post infection using CEA.hFcprotein and anti-hFc-PE secondary, plus anti-CD34-APC or by anti-CD34-PEantibodies alone. Cells were then expanded further using ×10 donormismatched irradiated PB MC feeders at a 1:20-1:200 ratio in RPMI+10%FCS with the addition of 1 μg/ml PHA and 200 IU/ml IL-2. After 14 daysthe cells were stained as previous and stored ready for assay.

Functionality assays were performed by mixing CoStAR positive ornegative cells with wild-type or OKT3 engineered CEA-Positive LoVo orLS174T cells. Briefly, T-cells were mixed with LoVo cells at varyingratios in 96-well plates and IFNγ or IL-2 measured by ELISA. Theremaining cells were incubated with 1:10 dilution of WST-1 reagent(Sigma, UK) for 30 min before absorbance reading at 450 nm. %Cytotoxicity was determined using the followingequation=100−((Experimental reading−T-cells alone)/(tumor alone))×100.

Proliferation assays were performed by first loading T-cells with 10 μMeFluor450 proliferation dye (eBioscience, UK) for 10 min at 37° C. at aconcentration of 1×10⁷ cells/ml before incubating the cells in 5 volumesof cold T-cell media for 5 min on ice. Cells were then washedexcessively to remove unbound dye and added to cocultures containingtumor cells. Cells were removed at 2, 6 and 10 days, 1:200 dilution ofDRAQ7 added and the cells analysed using a MACSQuant cytometer andMACSQuantify software.

Cell counts for proliferation assays were performed by taking cells fromthe wells and staining with anti-CD2 PerCP eFluor710 antibody(eBioscience, UK) for 20 min in the dark, followed by DRAQ7 staining andcounts made using a MACSQuant analyser.

Results

Primary human T-cells were isolated from Buffy coats obtained fromcommercial suppliers (Lonza or NHSBT). T-cells were isolated byFicoll-mediated isolation and T-cell negative isolation kits (StemCellTechnologies). The isolated T-cells were activated with human T-cellactivation and expansion beads (Invitrogen, UK). Cells were incubatedwith concentrated lentiviral particles and expanded over a number ofdays. The lentivirus contained the DNA sequence of theMFE.CoStAR.2A.tCD34 construct (MFE23.scFv fused to full length humanCD28 co-expressed with truncated human CD34 via a 2A cleavage sequence).Successfully transduced cells were further expanded using irradiatedfeeders as outlined in materials and methods. Donor 1 transduction wasmeasured at 22.69% (17.15 CD34+/CoStAR+ plus 5.53% CD34−/CoStAR+), donor2 was measured at 20.73%, and donor 3 at 13.34%. Cells were enriched forCoStAR expression using anti-CD34 antibodies to obtain T-cellpopulations greater than 90% CoStAR positive.

To generate a physiologically relevant in vitro model to test the impactof CoStAR on T-cell activity, the non-transduced and transduced cellswere tested against the CEA+ tumor cell lines LoVo and LS174T. To enableactivation of the T-cells in response to the unmatched tumor lines weengineered the tumor cells to express an anti-CD3 single chain antibodyfragment anchored to the cell membrane by way of a synthetictransmembrane domain and split from the GFP marker gene using an IRESelement to visualise transduced cells using flow cytometry.

Single cell clones of LoVo and LS174T were generated from bulktransfectants. Non-transduced and CoStAR transduced T-cells were mixedat varying effector:target ratios with wild-type non-transduced orOKT3-engineered LS174T or LoVo cells. After 24 hours coculture media wastaken for IL-2 ELISA measurement. Activation dependent IL-2 secretionwas observed from both CoStAR+ and CoStAR− T-cell populations from threedonors in response to OKT3 engineered LS174T cells with only backgroundIL-2 secretion seen from transduced and non-transduced T-cells inresponse to un-engineered tumor cells (FIGS. 3A-3C). CoStAR enhancedIL-2 secretion towards OKT3 engineered tumor cells was found in allthree donors tested. The effect was most evident at E:T ratios of 8:1and 16:1 and at higher E:T ratios IL-2 secretion was too low to measureaccurately. At lower effector to target ratios it appeared that IL-2secretion was saturating from non-transduced cells. These observationswere repeated in LoVo cells with two of the three donors tested againstLS174T with similar results (FIGS. 3D & E).

To determine the impact of CoStAR on T-cell expansion, transduced ornon-transduced T-cells were mixed with wild-type or OKT3-GFP engineeredLoVo cells the number of total cells after 3 days was counted. CoStARenhanced survival and/or proliferation of engineered T-cells in responseto LoVo-OKT3 but not wild-type LoVo cells in the presence of IL-2 (FIG.4A) and absence of IL-2 (FIG. 4B). To further investigate thisphenomenon, cell proliferation analysis was performed in T-cells fromtwo donors using proliferation dye to count the number of cell cycleseach population went through over 6 days (FIGS. 4C & D). A largerproportion of CoStAR engineered cells went through 5, 6 or 7proliferation cycles over 6 days compared to non-engineered cells inresponse to LoVo-OKT3, whereas CoStAR transduced and non-transducedcells went through an average of approximately 2 cycles over the sameduration in response to wild-type LoVo.

A variety of fusion receptors consisting of CD28 fused to an N-terminaladditional costimulatory domain were generated. Costimulatory domainsobtained from: CD137, CD2, CD29, CD134, CD150, CD40, GITR and thesignalling domain from the IL-2 receptor γ-chain (IL-2Ry) were chosen. Areceptor as close to that used in previous studies of induciblecostimulation was included. This receptor designated CD28(IEV) istruncated such that the C-terminal motif of CD28 is the amino acid triad‘IEV’. Sequences were generated de novo by Genewiz and cloned into alentiviral vector under an EF1α promoter along with a CD34 marker geneseparated from the fusion CoStAR by a 2A self-cleaving peptide. PrimaryCD8+ T-cells were isolated using EasySep beads (StemCell Technologies)and activated with anti-CD3/anti-CD28 activation/expansion Dynabeadsbefore addition of lentiviral particles. Following a short expansionperiod the cells were mixed with LoVo or LoVo-OKT3 cells, with theinclusion of anti-CD107a antibodies and brefeldin and monensin, andfollowing a 16 hour incubation were fixed and stained with antibodies tothe marker gene (CD34) as well as antibodies to IL-2, IFNγ and Bcl-xL.Analysis was performed using a MACSQuant analyser and MACSQuantifysoftware. FIG. 5 shows the IL-2 response from CD34− (CoStARnon-transduced) and CD34+(CoStAR transduced). Statistical analysisdemonstrated that all receptors tested induced a significant increase inthe proportion of cells producing IL-2 when harboring the variant CoStARreceptors. Three other read outs were concurrently measured: IFNγ, acytokine released under normal signal 1 conditions but enhanced bycostimulation; CD107a, a marker of degranulation; and Bcl-xL, anantiapoptotic protein upregulated by costimulation. Engagement of CoStARenhanced all the effector functions analysed to varying degrees.CD28.CD2 and CD28.CD40 fusions receptors appeared to elicit the mostrobust response of all the receptors tested (See FIGS. 6A-6D)

Example 2

The effect of CD28 and CD28.CD40 based CoStARs on population basedcytokine secretion was compared. Primary T-cells from three donors weretransduced with either the CD28(IEV) truncated CoStAR, full length CD28CoStAR or CD28.CD40 CoStAR (having the full length CD28 as shown in SEQID NO. 10, but lacking the N terminal N and K residues) or leftnon-transduced. T-cells were enriched for CoStAR expression using theCD34 marker gene, and following expansion cells were mixed withLoVo-OKT3 cells and IL-2 secretion analysed by ELISA (See FIG. 7).Non-transduced cells on average produced 0.80 ng/ml IL-2, with CD28(IEV)and full length CD28 CoStAR producing 4.6 and 5.0 ng/ml IL-2respectively. However CD28.CD40 induced 29.0 ng/ml IL-2 on averageacross three donors thus demonstrating a clear benefit to incorporatingCD40 into the basic CD28-based CoStAR.

Next the effect of CoStAR on T-cell expansion was analysed. T-cells fromseven donors were transduced with either CD28 or CD28.CD40 CoStARs witheither an anti-CA125 (196-14) or anti-Folate receptor (MOV-19) scFv, oran anti-Folate receptor peptide (C7) antigen binding domain. Additionalcells were transduced with a CD28 CoStAR harboring an anti-CEA scFv as amismatched control. Cells were then mixed with CA125+/Folatereceptor+/CEA− cell line OvCAR3 engineered to express a membrane boundOKT3 (OvCAR-OKT3). T-cell counts were made after 7, 14 and 21 days, andfresh OvCAR-OKT3 added at days 7, and 14. Limited expansion of cellsharboring the anti-CA125 scFv was observed (mean fold expansion: CD28:15.1; CD28.CD40: 69.1), however cells targeting Folate receptor with anscFv did expand in both the CD28 and CD28.CD40 cohorts (mean foldexpansion: CD28: 186.7; CD28.CD40: 1295.0). More limited expansion wasseen when the C7 peptide was used to target the Folate receptor (meanfold expansion: CD28: 71.5; CD28.CD40: 28.0). The control CEA targetingreceptor demonstrated limited expansion (mean fold expansion: 28.0).

To better understand the synergy of signal 1 and signal 2 T-cells wereengineered with a murine constant domain modified TCR which recognizes aCEA peptide (691-699) in the context of HLA-A*02 as well as the CD28 orCD28.CD40 CoStAR targeted towards cell surface CEA protein. As a controlcells were also transduced with a CA125 specific CD28 CoStAR. TheT-cells were mixed with HLA-A*02+/CEA+H508 cells and cytokine productionanalysed by intracellular flow cytometry staining. Flow cytometricgating was performed using antibodies directed towards the murine TCRβconstant domain (marks the TCR engineered cells) as well as the DYKDDDDK(SEQ ID NO:14) epitope tag (marks the CoStAR engineered cells). Thus itwas possible to analyse the TCR−/CoStAR−, TCR+/CoStAR−, TCR−/CoStAR+ andTCR+/CoStAR+ cells in each coculture well. Cytokine production was thenplotted in each subpopulation in either the CD4+ or CD8+ T-cells (FIG.9). In CD4+ cells CD28.CD40 CoStAR enhanced CD137 and TNFα productionabove TCR stimulation alone, however the TCR response in CD4+ cells waspoor due to the dependency of the TCR on CD8. In CD8+ cells there wasmore robust effector activity with IL-2 and CD107a in particular showinga stronger induction in the CD28.CD40 CoStAR groups. To better comparethe receptors the effector activity in just the TCR+/CoStAR+ groups wasplotted in CD4+ and CD8+ cells (FIGS. 10A-10B). In CD4+ cells inductionof CD137 was significantly enhanced by CD28.CD40 compared to either CEAor mismatched targeting CD28 CoStAR. In CD8+ cells CD137 induction wassignificantly increased compared to either CEA or mismatched targetingCD28 CoStAR, whereas CD107a induction was increased compared to thecontrol CoStAR. Thus CD28.CD40 shows enhanced effector activity across abroad range of models and effector activities.

Example 3

To evaluate costimulation by CD40 bearing CoStARs, primary human T-cellswere mock transduced or transduced with MFE23.CD28 or MFE23.CD28.CD40CoStAR, each harboring a CD34 marker gene separated by a 2A cleavagepeptide. MFE23 is a single chain Fv antibody that has a high affinityfor carcinoembryonic antigen (CEA). Following in vitro culture cellswere enriched for CD34 using MACS™ paramagnetic selection reagents(Miltenyi Biotech) and then the cells expanded in number usingirradiated feeder cells. MFE23.CD28 CoStAR strongly mediated expansionof CD34⁺ T cells, and MFE23.CD28.CD40 CoStAR further enhanced expansion(FIG. 11).

To evaluate costimulatory activity and persistence, T cells mocktransduced or transfected with MFE23.CD28 or MFE23.CD28.CD40 werecocultured with LoVo-OKT3 cells at an 8:1 effector:target ratio in thepresence (200 IU/ml) or absence of exogenous IL-2. At days 1, 4, 7, 11and 18 cells were taken and the number of viable T-cells enumerated byusing anti-CD2 reagents on a MACSQuant flow cytometer. In the absence ofstimulation by tumor and IL-2, cells declined in number as would beexpected (FIG. 12A). In the absence of stimulation but presence of IL-2there was a more apparent survival of the cells, but no specific growth(FIG. 12B). In the presence of tumor, but absence of IL-2 mock cells didnot show specific survival. MFE23.CD28 CoStAR mediated an apparentdoubling in expansion over the first four days followed by decline.MFE23.CD28.CD40 mediated a greater expansion up to day 7 followed by asteady decline (FIG. 12C). Under the same conditions but in the presenceof IL-2 both mock and MFE23.CD28 transduced cells demonstrated a 20-foldexpansion over 18 days, whereas MFE23.CD28.CD40 cells expanded by over60-fold (FIG. 12D). Thus CD28.CD40 based receptors demonstrated superiorexpansion and survival under conditions of stimulation both in thepresence and absence of exogenous IL-2.

Mock transduced and T cells transduced with MFE23.CD28 orMFE23.CD28.CD40 CoStARs were then tested for cytokine production. Beadarray analysis was performed on supernatants obtained from T-cell/tumorcocultures. Engineered T-cells were incubated at a 1:1 effector:targetratio with LoVo-OKT3 cells for 24 hours and supernatant collected.Conditioned supernatant was also collected from an equal number ofT-cells alone, or LoVo-OKT3 cells alone. Production of IL-2, IFN-γ,TNFα, IL-4, IL-5, IL-13, IL-17A, IL-17F, IL-22, IL-6, IL-10, IL-9, andIL-21 was analysed using a Legendplex™ Human TH1/TH2 cytokine panel(Biolegend) (FIGS. 13A-13M). Cytokines were either very low orundetectable in media from T-cells or tumor alone. However whencocultured with tumor cytokine production was enhanced. MFE23.CD28enhanced production of IL-2, IL-5, IL-17A/17F, IL-10, IL-9 and IL-21compared to mock. However, MFE23.CD28.CD40 also enhanced production ofTNFα, IL-13 and IL-22. MFE23.CD28.CD40 also enhanced the production of anumber of cytokines greater than that elicited by MFE23.CD28 (IL-2, IL-9and IL-17F), but also reduced the production of some cytokines below thelevels seen with MFE23.CD28 (IL-5 and IL-10). Together this datademonstrates that addition of CD40 to CD28-based Costimulatory receptorsenhances and/or modulates their specific activity with respect tocytokine production.

Mock transduced and T cells transduced with MFE23.CD28 orMFE23.CD28.CD40 CoStARs were further tested for chemokine production.Production of IL-8 (CXCL8), IP-10 (CSCL10), Eotaxin (CCL11), TARC(CCL17), MCP-1 (CCL2), RANTES (CCL5), MIP-1a (CCL3), MIG (CXCL9), ENA-78(CXCL5), MIP-3a (CCL20), GROα (CXCL1), I-TAC (CXCL11), and MEP-1β (CCL4)was analysed using a Legendplex™ Human Pro inflammatory chemokine panel.(FIGS. 14A-14M). Chemokines were either very low or undetectable inmedia from T-cells alone. When cocultured with tumor, chemokineproduction was enhanced. MFE23.CD28 enhanced production of CXCL5,CXCL10, CXCL11, CCL17 and CCL20 compared to mock. However,MFE23.CD28.CD40 enhanced production of CCL2, CXCL1 and CXCL9.MFE23.CD28.CD40 also further enhanced the production of certaincytokines to a greater amount than that elicited by MFE23.CD28 (CXCL1,CXCL9, CXCL10, CXCL11, CCL17, CCL2, CXCL9, CCL5 and CCL20), whilereducing the production of some cytokines below the levels seen withMFE23.CD28 (CCL4). Together this data demonstrates that addition of CD40to CD28-based Costimulatory receptors enhances and/or modulates theirspecific activity with respect to chemokine production.

CoStARs were tested for functional activity against cancer targets.Cells were transduced with CD28 or CD28.CD40 CoStARs engineered with anscFv binding domain specific for FolR or CA125 (scFv MOV19 and scFv196-14 respectively). Human folate receptor alpha (FolR) represents asuitable target for a number of tumors including ovarian, head and neck,renal and lung and CA125 represents an alternative target for ovariancancer. Primary human T-cells from six healthy donors were engineeredwith either 196-14.CD28, 196-14.CD28.CD40, MOV19.CD28 or MOV19.CD28.CD40receptors, all harboring a DYKDDDDK epitope tag for detection.Transduced cells were mixed with FolR+/CA125+ OvCAR-OKT3 cells beforeanalysis of effector activity using intracellular staining in theepitope tag positive and negative populations. Specific enhancement ofeffector activity determined by production of IL-2 (FIGS. 15A and 15B),TNFα (FIGS. 15C and 15D), CD137 (FIGS. 15E and 15F), and BCL-xL (FIGS.15G and 15H) was observed in CD28 and CD28.CD40 engineered cellscompared to mock transduce cells in response to both CA125 and FolR,although specific BCL-xL induction by MOV19.CD28 was not substantial ascompared to MOV19.CD28.CD40.

Mock transduced TILs or TILs engineered with MOV19.CD28.CD40 CoStAR wereevaluated for expansion and CD137 production stimulated by patientmatched tumor digest (FIGS. 16A-16F). Three donor tumors were testedwhich displayed varying levels of FolR on the digest, ranging fromnegative (FIG. 16A), low expression (FIG. 16B) to high expression (FIG.16C). Mock and CoStAR negative TIL in the CoStAR engineered populationsof TIL matched for the FolR negative digest demonstrated similar levelsof CD137 upregulation following tumor coculture which was not enhancedby the presence of CoStAR (FIG. 16D). In the TIL exposed to FolR lowexpressing digest there was an enhancement in activity in the CoStAR+cells compared to CoStAR−, with CD137 expression increasing from <10%to >20% (FIG. 16E). In the TIL exposed to FolR high expressing tumordigest there was an increase in activity from around 20% in the CoStAR−population, up to approximately 50% in the CoStAR+ population (FIG.16F).

A FolR targeting CoStAR was examined for enhancement of effectorfunctions. MOV19.CD28.CD40 enhanced CD137 expression from ˜20% to ˜50%(FIG. 17A), TNFα production from 10% to 15% (FIG. 17B) and IL-2production from 2% to 5% (FIG. 17C) in response to FolR+ tumor digest.

CoStAR mediated stimulation by soluble ligand was also examined. T-cellsfrom three healthy donors were engineered with MOV19.CD28 orMOV19.CD28.CD40 CoStAR and activated with either immobilised OKT3,providing stimulation in the absence of FolR, or with OvCAR-OKT3, toprovide TCR and CoStAR activity. Bcl-XL activity was increased frombetween 10 and 20% across the three donors following OKT3 stimulation(FIG. 18A) whereas IL-2 was increased between 0 and 12% (FIG. 18B) andTNFα increased between 0 and 20% (FIG. 18C). The presence of exogenoussoluble FolR did not enhance any of these particular effector functions.In the presence of OvCAR-OKT3 Bcl-XL induction was enhanced by ˜20% inCD28 CoStAR and by ˜35% in CD28.CD40 CoStAR (FIG. 18D), IL-2 inductionwas enhanced by ˜20% in CD28 CoStAR and 30-50% in CD28.CD40 CoStAR (FIG.18E) and TNFα production was enhanced by 20-30% in CD28 CoStAR and25-50% in CD28.CD40 CoStAR (FIG. 18F). Exogenous soluble FolR did nothave an inhibitory effect on any of these effector functions.

Example 4 Materials and Methods

Construct design—The MFE23, MOV19 and 196-14 CoStAR constructs includean MFE23 (CEA specific), MOV19 (Folate receptor a specific) or 196-14(CA125 specific) derived single chain antibody fragment nucleotidesequence with an oncostatin M1 leader sequence fused to a costimulatorydomain. The costimulatory domains contain an extracellular spacer regionand transmembrane domain derived from human CD8 or CD28 and a signallingdomain of either CD28, CD2 or CD137 and/or wild-type or mutant CD40variants. Some CoStARs detailed herein comprise a human PD1extracellular domain fused to CD28 and CD40. Receptors were cloned witha P2A cleavage sequence and a truncated form of human CD34 to permitdetection of transduced cells. The CoStAR nucleotide sequence was codonoptimised and gene synthesised by Genewiz Inc. The constructs werecloned into a third generation lentiviral vector.

Peripheral blood mononuclear cells were isolated from normal healthydonors before activation for 24 hours with T-cell activation andexpansion beads (Invitrogen) according to the manufacturer'sinstructions before addition of lentiviral supernatants.

Cell transduction was assessed 96 hours post infection using CEA.hFcprotein (R&D Systems) and anti-hFc-PE secondary, plus anti-CD34-APC orby anti-CD34-PE antibodies alone. Cells were then expanded further using×10 donor mismatched irradiated PBMC feeders at a 1:20-1:200 ratio inRPMI+10% FCS with the addition of 30 ng/ml OKT3 and 200 IU/ml IL-2.After 14 days the cells were stained as previous and stored ready forassay.

Functionality assays were performed by mixing CoStAR positive ornegative cells with wild-type or OKT3 engineered CEA-Positive LoVocells. Briefly, T-cells were mixed with LoVo cells at varying ratios in96-well plates. For flow analysis cocultures were incubated withBrefeldin and monensin and anti-CD107a antibodies for 16 hours followingwhich cells were stained with Fixable Viability Dye ef450(eBiosciences), fixed with 4% paraformaldehyde and then permeabilisedusing Fix/Perm wash buffer (BD Biosciences). Cells were then stainedwith anti-CD34 or anti DYKDDDDK antibodies to differentiate between theCoStAR+ and CoStAR-populations, anti-IL-2, anti-TNFα and anti-IFNγantibodies (Biolegend). For soluble analyte analysis supernatants werecollected for analysis by ELISA, cytokine bead array (LEGENDPLEX™ HumanTh Cytokine Panel (12-plex)) or chemokine bead array (LEGENDPLEX™ HumanProinflammatory Chemokine Panel (13-plex).

Proliferation assays were performed by mixing T-cells and tumor cells atan 8:1 effector:target ratio in complete T-cell media (TCM: RPMIsupplemented with 10% FCS, 0.01 M HEPES and 1% Penicillin/streptomycin,50 mM 3-mercaptoethanol) in the presence or absence of IL-2. Cell countswere made at indicated time points and fresh tumor cells were added inrestimulation assays at a final E:T of 8:1. Cell counts forproliferation assays were performed by taking cells from the wells andstaining with anti-CD2 PerCP eFluor710 antibody (eBioscience, UK) for 20min in the dark, followed by DRAQ7 staining and counts made using aMACSQuant analyser.

Example 5

To evaluate the in vivo anti-tumor activity of T cells transduced withCD40 bearing CoStARs, primary human T-cells are mock transduced ortransduced with MOV19.CD28.CD40 CoStAR construct followed by in vitroexpansion and cryopreservation. MOV19 is a single chain Fv antibody thathas a high affinity for Folate Receptor alpha (FOLR1). Immunocompromisedmice are implanted with an established ovarian cancer cell line (A2870,OVCAR-5, OVCAR-8 or SK-OV-3), which is allowed to grow in the animal forfew days. Mice are subsequently staged according to their tumor burden,and finally injected with the mock transduced T cells or MOV19.CD28.CD40transduced T cells. Shortly after the T cell dosing, some of the miceare injected with intravenous IL-2 (5 μg IL-2, Q2Dx7) to support theengraftment and initial expansion of T cells. The final study designcontains 5 groups (each one containing 5 mice): PBS (no cells dosed),mock transduced T cells, mock transduced T cells with IL-2supplementation, MOV19.CD28.CD40 transduced T cells and MOV19.CD28.CD40transduced T cells with IL-2 supplementation. Tumor growth and micesurvival is monitored on weekly basis for a total of 40 days.

Mice administered with MOV19.CD28.CD40 transduced cells show bettertumor control and prolonged survival compared to the mock transducedgroups, whether or not supplemented with IL-2. This data demonstratesthe ability of the CoStAR platform to improve in vivo the T cellanti-tumor response and also illustrates how this improved response isindependent of the presence of exogenous IL-2.

Example 6

The example relates to identification of key components of CoStAR suchas, but not limited to, PD-1, MFE23, CD40 combined with anothercomponent, a spacer, a CD40 mutant and/or a CD28 mutant.

Virus production was carried out by CaCl₂) transfection of HEK293Tcells. CD34 (a marker gene) expression was determined by titration withJRT3 cells.

An experimental design for outgrowth in healthy donors was as follows:Day 0 was T cell isolation from frozen PBMCs. Day 0 was also activationwith Dynabeads. Day 2 was transduction by spinoculation. Day 5 was beadremoval. Day 8 was measuring viability and transduction rate. Day 8 wasalso post activation (before REP), Days 13-15 was freezing.

CD34 expression after magnetic enrichment, before REP, was measuredbefore sort and in positive and negative fractions after sort. Healthydonors were activated with Dynabeads and transduced (spinoculation, MOI5) with CD40 CoStAR constructs or MOCK. Cells were then magneticallyenriched for their CD34 expression and analyzed by flow cytometry(Novocyte) before and after sort

An experimental design in healthy donors included the outgrowth asdescribed above as well as REP: Day −2 was transduced T cells thawing,Day −1 was magnetic CD34 enrichment, Day 0 was REP with G-Rex, Day 5-6was changing medium, Day 11-12 was measuring viability and transductionrate and freezing.

The majority of CD40 CoStAR modified T cells were enriched in CD4 afterCD34 enrichment and REP (FIG. 20). CD4 and CD8 T cell phenotypes wereassessed 10-11 days after REP using anti-human CD4-PerCP-eF710,anti-human CD8-PE-Cy7, and anti-human CD3-FITC. Analysis was performedby flow cytometry (Novocyte) and data were analyzed using NovoExpress1.5.0 software with the following gating strategy: live/dead exclusion,single cells, CD3+ cells, CD4+ cells or CD8+ cells.

CoStAR modified CD4 T cells were highly transduced compared to the CD8population. Healthy donors were activated with Dynabeads and transduced(spinoculation, MOI 5) with CD40 CoStAR constructs or MOCK. Cells werethen magnetically enriched for their CD34 expression and expandedfollowing the rapid expansion protocol (REP). Surface expression of themarker gene CD34 on CD4 and CD8 T cells, was assessed 10-11 days afterREP using anti-human CD34-PE associated with anti-human CD4-PerCP-eF710,anti-human CD8-PE-Cy7, and anti-human CD3-FITC. Analysis was performedby flow cytometry (Novocyte) and data was analyzed using NovoExpress1.5.0 software with the following gating strategy: live/dead exclusion,single cells, CD3+ cells, CD34+ cells among CD4+ cells or CD8+ cells.

Example 7

CoStARs composed of an extracellular checkpoint binding domain fused toa CD40 costimulatory domain could convert an inhibitory signal into anactivating signal upon engagement of the CoStAR. To test theapplicability of such receptors we generated PD1-fusion CoStARs based onthe descriptions outlined in Ankri et al. J Immunol 2013; 191:4121-4129and Prosser et al. Molecular Immunology 51 (2012) 263-272, but with theaddition of CD40 to the signalling domain (FIG. 21A). Primary humanT-cells isolated from healthy donors were activated with CD3/CD28Dynabeads and transduced with the indicated PD1 fusion CoStAR receptorsat an MOI=5, or an MFE23.CD28.CD40 CoStAR (positive control) or mocktransduced (negative control). Transduced T-cells were enriched usingCD34 microbeads and expanded via a rapid expansion protocol usingirradiated feeder cells before banking. After thaw, cells were restedfor 3-4 days in complete RPMI supplemented with IL-2. The viability andabsolute count were assessed after overnight IL-2 starvation usingDRAQ-7 (1:200) by flow cytometry (Novocyte) and data were analysed usingthe NovoExpress 1.5.0 software. Transduced T cells were cocultured inthe absence of IL-2 with LoVo (CCL-229TM) or LoVo.OKT3.GFP tumor cellsat 8:1 effector to target ratio. After 24 hours, supernatants werecollected and frozen. LoVo and LoVo.OKT3.GFP naturally express CEA andPD-L1 on their surface, conferring signal 2 through the CoStAR alone(LoVo) or associated with signal 1 (LoVo.OKT3.GFP) to the transduced Tcells. Cocultures were performed in triplicate and correspondingnegative (T cells alone, tumor cells alone) and positive (PMA+ionomycin)controls were included in the experiment. Secreted IL-2 and IFN-γ weredetected by ELISA and the absorbance was measured using a FLUOstar Omegamicroplate reader and subsequently analysed with the Omega MARS 3.42 R5software. Each symbol is the average triplicate value for each donor(FIG. 21C). MFE23.CD28.CD40 (CTP194) expressing cells produced onaverage approximately 4000 pg/ml IL-2 in the presence of LoVo-OKT3cells, whereas production from PD1 fusions was <1000 pg/ml. Analysis ofIFNγ secretion also demonstrated enhanced production of this cytokinefrom MFE23.CD28.CD40 engineered cells compared to mock, however noenhancement in production from cells harboring the PD1 fusions wasobserved. Next, we assessed the ability of PD1-fusion CoStARs to mediateT-cell survival in the presence of repeated tumor challenge (FIG. 22).To this end CoStAR or mock transduced T-cells were mixed at 8:1 E:Tratio with LoVo-OKT3 cells at day 0 and 7 and counts and checkpointexpression phenotyping made at day 6-8 and 14-15 (FIGS. 23A-23C). FIG.22 shows the fold expansion of cells over the duration of theexperiment, with mock transduced cells dropping in number throughout theexperiment; conversely MFE23.CD28.CD40 (CTP194) engineered cellsexpanded upon serial stimulation with tumor up to 12-fold by day 14.Although PD1 fusion CoStARs did not demonstrate a similar degree ofexpansion to CTP194 engineered cells, the degree of T-cell death was notas great as mock engineered cells suggesting that the PD1 domain canmediate some degree of T-cell survival. Checkpoint expression (LAG3, PD1and TIM3) was also assessed in the CD4+ and CD8+ cells in both CD34− andCD34+ populations and is shown at days 6-8 and 14-15 on FIGS. 23A-23C.LAG3 was found to be relatively low (typically <20%) at day 6-8 in CD4+and CD8+ cells (albeit more variably in CD8+ cells) with no obviousdifference between cells harboring the different receptors or mocktransduced. However, at day 14-15 we observed lower LAG3 expression incells harboring the PD1 or MFE23 based CoStAR compared to mockengineered cells. PD1 expression was more difficult to assess as PD1 asa component of the CoStAR could not be separated from endogenousexpression. In CD4+ cells we observed lower PD1 expression inMFE23.CD28.CD40 engineered cells compared to mock transduced, an effectwhich was more obvious at day 14-15. Expression of TIM3 mirrored LAG3expression in CD4+ and CD8+ cells at day 6-8, whereas at day 14-15 TIM3expression was generally low, however we did observe a high expressionof TIM3 in mock transduced cells at day 14-15 (˜80% of cells), which waslower in PD1-fusion CoStAR cells, but <20% in cells harboringMFE23.CD28.CD40. In summary, CoStARs consisting of an antigenrecognition domain which inverts signals, such as PD1, are functionalbut do not perform as well in cytokine release or expansion assays ascells harboring CoStAR with an scFv-based antigen recognition domain.PD1-fusion receptors can also modulate checkpoint expression compared tomock engineered cells as well.

Next we sought to understand how CD40 may operate as a single componentof the CoStAR, or in combination with costimulatory domains other thanCD28. To control for the effect of receptor oligomerisation andstoichiometry we used a base CD8 transmembrane domain for each fusionand compared with the control MFE23.CD28.CD40 receptor (FIG. 24A). Thefirst receptor consists of a CD28.CD40 signalling domain with CD8extracellular and transmembrane domain (CTP190), CD2.CD40 signallingdomain with CD8 extracellular and transmembrane domain (CTP191), orCD137.CD40 signalling domain with CD8 extracellular and transmembranedomain (CTP192). Additionally, a receptor consisting of CD40 alone(CTP193) was also generated. Flow cytometric analysis of transducedcells showed that expression of these receptors did not correlate wellwith CD34 marker gene expression, suggesting that the structural formatsdid not permit efficient surface expression (FIG. 24A, lower leftpanel). Nonetheless we conducted functionality assays and showed thatthe CD28.CD40, CD137.CD40 and CD40 receptors could mediate enhanced IL-2secretion compared to mock transduced cells in LoVo-OKT3 cocultures,whereas IFNγ secretion was lower than from mock transduced cells, andMFE23.CD28.CD40 engineered cells (FIG. 24B). Analysis of expansion inthe presence of LoVo-OKT3 cells (FIG. 25) demonstrated thatMFE23.CD28.CD40 based receptors mediated optimal expansion with thoseharboring the native CD28 extracellular and transmembrane domain(CTP194) outperforming those with CD8 derived transmembrane domains(CTP190). The fusion of CD137 and CD40 maintained T-cell numbersthroughout the experiment with no associated expansion, whereasreceptors consisting of CD2.CD40 or CD40 alone did not support long termsurvival. Akin to an effect seen with mock transduced cells. Phenotypicanalysis of cells at day 6-8 and 14-15 was also conducted (FIGS.26A-26C). LAG3 was very low on CD4 cells at day 6-8, and <20% on CD8+cells. At day 14-15 LAG3 was present on ˜50% of CD4+ mock cells but <20%of engineered cells (data unavailable for some receptors due toinsufficient cell numbers to analyse). PD1 expression was again <20% onCD4+ and CD8+ cells at both time points analysed except for the CD2.CD40CTP191 engineered cells at day 6-8 and mock transduced cells at thelater time point. TIM3 expression was generally low in CD4+ cells atboth timepoints analysed, but higher in CD8+ cells, in particular inCD137.CD40 (CTP192) and CD40 (CTP193) engineered cells. Finally, mocktransduced cells displayed >70% expression of TIM3 at day 14-15 whereasthose harboring either CD28.CD40 CoStAR had ˜20% expression. In summaryCoStARs containing any combination of costimulatory domain tested withCD40 can modulate checkpoint expression, but this effect is mostapparent in combination with CD28, and better than when CD40 is used asa sole signaling component.

Next we sought to investigate the effect of specific mutations withinthe MFE23.CD28.CD40 construct, in an effort to understand how differentsignaling components are responsible for the optimal activity of theCTP194 MFE23.CD28.CD40 receptor. To this end we introduced mutationsinto a known TRAF2 binding motif (SVQE-AVQA) (CTP195), a TRAF2/3 bindingdomain (PVQET-AVAEA) (CTP196), and a TRAF6 binding domain(PQEINF-AQAINF) (CTP197). We also introduce point mutations to introducea polymorphic variant of CD40 which has been shown to have enhancedactivity in B-cells (P227A) (CTP198) and a Q263A mutation which has beenshown to affect TRAF3 binding (Leo et al. J Biol Chem. 1999) (CTP199).Finally the cohort of receptors was completed by cloning of a CoStARwith a triplicated CD40 intracellular domain (CTP200) (FIGS. 27A-27B).As previously, primary human T-cells were transduced with lentiviralvectors encoding these receptors, enriched using CD34 microbeads andfrozen prior to experimentation. Expression levels of CD34 were onaverage between 60 and 70% (FIG. 27A). Transduced or mock transducedcells were mixed with LoVo-OKT3 cells, and IL-2 and IFNγ measured byELISA after 24 h (FIG. 29B). IL-2 production from mock transduced cellswas below the level of detection. IL-2 production from the controlCTP194 receptor was approximately 4000 pg/ml, as was production fromCTP195 harboring the SVQE-AVQA mutations, and cells harboring the CTP198receptor with the P227A polymorphism. Cells expressing the TRAF6 bindingmutations PQEINF-AQAINF, or the TRAF3 binding mutation Q263A, as well ascells expressing the triplicated CD40 motif, all demonstrated moderatereductions in IL-2 production. However, cells expressing CTP196containing the TRAF2/3 binding motif mutations PVQET-AVAEA displayed aconsiderable reduction in IL-2 production. This cytokine reduction wasalso observed when IFNγ was measured, with all receptors producing >30ng/ml IFNγ, except CTP196 which produced approximately 10 ng/ml, similarto mock transduced cells.

Next we assessed the impact of these different CD40 signalling domainmutations on the ability to support repeat stimulation (FIG. 28). Tothis end mock or transduced T-cells were mixed with LoVo-OKT3 cells atan 8:1 E:T ratio and counts made at day 6-8 and 14-15 across threedifferent donors. CTP194 expressing cells expanded approximatelyfour-fold by the first time point, and upon restimulation expandedto >10-fold. Cells expressing the TRAF2 binding mutation CTP195, TRAF6binding mutation CTP197 or P227A polymorphism (CTP198), had moderatereductions in ability to support restimulation, whereas cells expressingthe C263A TRAF3 binding mutant, or triplicated CD40 binding domain werefurther disabled in their ability to expand cells. Strikingly, cellsexpressing the TRAF2/3 binding mutant CTP196 were profoundly impacted intheir ability to support repeat stimulation. Phenotypic analysis of cellexpressing these different mutations was also conducted (FIGS. 29A-29B).No clear differences were seen in the relative expression of LAG3 inCD4+ or CD8+ cells at days 6-8 between transduced and non-transducedcells, However mock transduced cells had higher LAG3 expression at days14-15 compared to CD4+ cells expressing any of the CoStARs. Nodifferences were observed with regards LAG3 expression in CD8+ cells atdays 14-15. PD1 expression was found to be <20% on average at days 6-8for all receptor engineered CD4+ cells, with higher expression in mockengineered cells. Interestingly we also observed elevated PD1 expressionin CD4+ and CD8+ cells expressing the TRAF2/3 motif mutant CTP196 atdays 6-8, and in CD4+ cells at days 14-15. TIM3 expression was found tobe lower than 20% on average in all CD4+ cell groups at both time pointsanalysed. Expression was generally more variable in CD8+ cells at thefirst time point, with an average of approximately 30%, althoughslightly higher in cells expressing CTP196. At days 14-15 mocktransduced cells had considerably higher TIM3 expression than transducedcells and cells expressing CTP196 had approximately twice as much TIM3expression than cells from other groups.

The final group of receptors tested were those containing CD28 harboringmutations to the YMNM and PYAP cytoplasmic motifs which are critical foractivating signal cascades involving PKCθ, PI3k and Lck amongst others(Esensten et al. 2016). CTP201 contains a PYAP-AYAA mutation, whereasCTP202 contains a YMNM-FMNM mutation. We also included a receptor withan extended IgG4 hinge into this cohort to establish whether CoStARscontaining a longer linker domain maintain functionality (CTP203) (FIG.30A). As previously, cells were transduced and enriched with CD34microbeads. Expression after sort and expansion was found to beapproximately 60% for the wild-type control and CTP201 and CTP202receptor expressing cells, but lower at 30% for the CTP203 IgG4 hingedomain receptor expressing cells (FIG. 30A, lower panel). IL-2production from mock or transduced T-cells was assessed followingcoculture with LoVo-OKT3 cells (FIG. 30B). IL-2 from CTP194 expressingcells was approximately 4000 pg/ml and lower for the CD28 mutantreceptors, both being approximately 2500 pg/ml. However, IL-2 from IgG4hinge receptor expressing cells was lower at approximately 1000 pg/ml.As a control IL-2 from mock transduced cells was below the lower levelof detection. We also measured IFNγ from the same cells (FIG. 30B). IFNγfrom CTP194 was approximately 1000 pg/ml, as was IFNγ from CTP201 cellsharboring the PYAP-AYAA CD28 mutation. We observed enhanced IFNγsecretion from cells expressing the YMNM-FMNM mutation. Within thiscohort of receptors, the production of IFNγ was highest from cellsexpressing the CTP203 IgG4 hinge receptor.

Next we analysed the ability to support expansion following tumorrestimulation with this cohort of receptors (FIG. 31). Cells expressingMFE23.CD28.CD40 expanded approximately 10-fold following two rounds ofstimulation with LoVo-OKT3 cells. We observed that mutations to the CD28signalling domain had a profound effect on the ability of cells toexpand over two rounds of stimulation, as did use of receptorscontaining the IgG4 hinge/spacer domain. Mock cells dropped in numberover the two rounds of stimulation. Phenotypic analysis of cells afterthe first stimulation (6-8 days) revealed no obvious differences in LAG3expression within CD4+ or CD8+ cells with the former expressingapproximately 10% expression on average, and the latter 20% (FIGS.32A-32C). However, LAG3 expression was higher in CD4+ mock cells at days14-15 at approximately 50%+, compared to an average of 10% or lower intransduced cells, and was also higher in CD8+ mock cells compared totransduced cells. PD1 expression analysis revealed approximately 10%expression in MFE23.CD28.CD40, or CD28 mutant CD4+ cells at the firsttime point, whereas cells expressing the IgG4 receptor had >20% PD1expression, as did mock transduced cells. At days 14-15 the differencewas greater still with 100% of mock CD4+ cells being PD1+. CD8+ cellsdemonstrated low PD1 positivity at both time points. Finally, no obviousdifference was seen in CD4 or CD8+ TIM3 expression at days 6-8, howeverat days 14-15 CD4+ cells expressing the IgG4 spacer domain receptorshowed higher PD1 positivity compared to cells expressing the control orCD28 mutant receptors, with a similar effect observed in CD8+ cells.

Example 8

Coculture assay set up. Effector (ie, Non-Td and Td) T cells were thawedone day prior to coculture, resuspended at 1×10⁶ cells/mL in TCM withoutIL-2, and incubated overnight at 37° C. with 5% CO₂. On the day ofcoculture, T cells and BA/F3 targets (ie, WT, OKT3, FOLR1, andOKT3-FOLR1) were collected and counted using a ViCELL BLU permanufacturer's instructions. Both Non-Td and Td T cells werepreincubated for 30 minutes at room temperature with a range of solFOLR1(ie, 0, 20, 60 and 200 ng/mL) concentrations that representconcentrations reported in ovarian cancer patient serum as well assupraphysiological levels. Following incubation, cells were coculturedwith either BA/F3 WT, OKT3, FOLR1, or OKT3-FOLR1 targets at thefollowing E:T ratios (3:1, 1:1, 1:3) overnight. Each condition wasperformed in duplicates. T cells stimulated with PMA/ionomycin as permanufacturer's instructions and unstimulated T cells served as positiveand negative controls, respectively. Following overnight, plates werecollected and centrifuged at 500×g for 3 minutes. 100 μL of supernatantwas collected from each well and stored at −80° C. prior to analysis ofcytokine content. The remaining cells in plates were then stained asdescribed below.

For proliferation coculture assays, T cells were first labeled withCellTrace™ Violet Dye on the day of coculture setup, according to themanufacturer's instructions. The labeled cells were then cultured for 5days with BA/F3 target cells (BA/F3, BA/F3-FOLR1, BA/F3-OKT3-FOLR1) at aE:T of 10:1.

Flow staining and analysis of coculture assay. After collectingsupernatant, pelleted cells were washed and labeled with 100 μLLive/Dead Fixable Near IR dye (prepared by adding 1 μL reconstituted dyeto 1 mL PBS) for 30 minutes at room temperature. All wash steps wereperformed by adding 100 μL stain buffer to each well, centrifuging at500×g for 3 minutes and decanting the supernatant. Following incubationwith the Live/Dead Dye, cells were washed and blocked with Fc block(1:50 dilution) for 15 minutes at room temperature following which 50 μLantibody cocktail was added to each well and incubated for 30 minutes at4° C. Cells were then washed and a volume of 100 μL BD Cytofix bufferwas added and cells were incubated for 20 minutes at 4° C. After fixing,cells were washed, reconstituted in 150 μL stain buffer, and stored in4° C. until analysis using a BD LSR Fortessa X-20.

Prior to cytometric analysis, plates were centrifuged at 500×g for 3minutes, and 25 μL counting beads (ie, 26000 beads) with 125 μL FACsstain buffer was added to each well. A total of 100 μL of the sample wasacquired from each well. The gating strategy was as follows:

Lymphocytes (forward scatter [FSC]—A vs side scatter [SSC]-A)

Single cells gate 1 (FSC-H vs FSC-A)

Single cells gate 2 (SSC-H vs SSC-A)

Viable cells (FSC-H vs Near Far IR APC-Cy7 dye)

Tumor vs T cells (anti-mouse CD45 BV785 vs anti-human CD45 BV650)

Activation markers 4-1BB (FSC-H vs anti-human 4-1BB BV421) and CD69(FSC-H vs anti-human CD69 BV711) gated specifically from T cells

Bead count from each well was recorded using the following gatingstrategy:

FSC-A vs SSC-A

SSC-H vs FITC

Analysis was performed using FlowJo (BD, version 10). Graphs wereplotted using GraphPad Prism 9.

Cytokine analysis. Supernatants collected from coculture assays asdescribed above were evaluated either neat or at 1:200 dilution withDiluent 2 from the MSD V-Plex Plus Proinflammatory Panel 1 kit from Mesoscale discovery (MSD). The assay was carried out according to themanufacturer's instructions and analysis performed using MSD discoveryworkbench software.

Expression of activation markers (4-1BB and CD69) and cytokineproduction (IL-2 and IFN gamma) respectively, from non-transduced (NTD)and anti-FOLR1 CoStAR modified T cells (CoStAR) from 3 healthy donorsco-cultured overnight with Ba/F3 targets. 4-1BB expression was higher inthe anti-FOLR1 CoStAR modified T cells than the NTD cells whereas CD69expression was similar in both cells (FIG. 34A). IL-2 and IFN gammaexpression was higher in the anti-FOLR1 coSTAR modified T cells than theNTD cells (FIG. 34B).

Tumor counts of Ba/F3 targets assessed by flow cytometry after overnightcoculture with NTD and CoStAR T cells were comparable in the anti-FOLR1CoStAR modified T cells and the NTD cells (FIG. 34C).

NTD and CoStAR T cell counts as well as proliferation assessed by flowcytometry after overnight or 5-day coculture with Ba/F3 targetsindicated that total cell counts and proliferation of both CD4 and CD8 Tcells were higher in the anti-FOLR1 CoStAR modified T cells than the NTDcells (FIG. 34D).

Expression of activation markers (4-1BB and CD69) from non-transduced(NTD) and anti-FOLR1 CoStAR modified T cells (CoStAR) from 3 healthydonors pre-incubated with increasing concentrations of soluble folatereceptor (sFOLR) and co-cultured overnight with Ba/F3 targets wascomparable in the anti-FOLR1 CoStAR modified T cells and the NTD cells,with the exception of increased expression of the anti-FOLR1 CoStARmodified T cells than the NTD cells FIGS. 25A and 25B).

Tumor counts of Ba/F3 targets assessed by flow cytometry after overnightcoculture with NTD and CoStAR T cells pre-incubated with increasingconcentrations of sFOLR were comparable in the anti-FOLR1 CoStARmodified T cells and the NTD cells (FIG. 35C).

Expression of cytokine production (IL-2) of NTD and CoStAR T cellspre-incubated with increasing concentrations of sFOLR assessed by flowcytometry after overnight coculture with Ba/F3 targets were comparablein the anti-FOLR1 CoStAR modified T cells and the NTD cells.

Expression of activation markers (4-1BB and CD69) from non-transduced(NTD) and anti-FOLR1 CoStAR modified T cells (CoStAR) from 3 healthydonors co-cultured overnight with Ba/F3 targets were comparable in theanti-FOLR1 CoStAR modified T cells and the NTD cells FIG. 36A).

Expression of cytokine production (IL-2), from non-transduced (NTD) andanti-FOLR1 CoStAR modified T cells (CoStAR) from 3 healthy donorsco-cultured overnight with Ba/F3 targets was increased in the anti-FOLR1CoStAR modified T cells as compared to the NTD cells (FIG. 36B).

Tumor counts of Ba/F3 targets assessed by flow cytometry after overnightcoculture with NTD and CoStAR T cells were comparable in the anti-FOLR1CoStAR modified T cells and the NTD cells (FIG. 36C).

NTD and CoStAR T cell counts assessed by flow cytometry after overnightor 5-day coculture with Ba/F3 targets was increased in the anti-FOLR1CoStAR modified T cells as compared to the NTD cells (FIG. 36D).

Example: 9

Production of CoStAR TIL

TIL from 6 ovarian tumors were liberated by digestion and cultured in3000 U IL-2. Transduction with a 3rd generation lentiviral vectorencoding a CoStAR molecule with and scFv targeting human FOLR1, linker,full length CD28 fused to truncated CD40 cytoplasmic domain was carriedout at an MOI of 5, both 48 h and 72 h after tumor digestion.

Flow cytometric analysis was used to determine the frequency of CD4 andCD8 T-cells expressing the CoStAR Molecule using an anti-idiotypeantibody for surface detection. About 20% to 70% of CD4 and CD8 T-cellsexpressed the CoStAR molecule (FIG. 37A).

Flow cytometric surface staining analysis was used to determine thefrequency of cells expressing TCRαβ and TCRγδ. About 100% of CD3+ cellsexpressed TCRαβ and considerably fewer CD3+ cells (close to zero)expressed TCRγδ (FIG. 37C).

CoStAR modified TIL from 6 ovarian tumors were co-cultured withautologous digest overnight in the presence of brefeldin A. Thefrequency of cells expressing IL-2 or TNFα was assessed the followingday by flow cytometry. The frequency of TIL reacting to autologousdigest is enhanced by the CoStAR molecule (FIG. 38A).

CoStAR modified TIL from 6 ovarian tumors were co-cultured withautologous digest and supernatant assessed for cytokine release. CoStARmodified cells had increased effector functions as demonstrated byincreased IFNγ, TNFα and IL-13 release. Maximal levels of thesemolecules was similar in response to stimulation with PMA (Phorbol12-myristate 13-acetate) and ionomycin (FIG. 38B).

CoStAR modified TIL from 5 ovarian tumors were co-cultured with BA/F3cells or BA/F3 cells engineered to express OKT3, FOLR or both. Cytokinesecretion of non-modified and CoStAR modified TIL was equivalent whenco-cultured with non-modified BA/F3 or BA/F3 expressing OKT3 alone orFOLR1 alone. CoStAR modified TIL secreted increased levels of cytokinesIL-2 and IFNγ when co-cultured with BA/F3 modified to express both FOLR1and OKT3 (FIG. 39A)

CoStAR modified TIL from 5 ovarian tumors were co-cultured with BA/F3cells or BA/F3 cells engineered to express OKT3, FOLR or both.Cytotoxicity towards BA/F3 target cells was assessed via cell counts,determined by flow cytometric analysis of mouse CD45. Non-modified andCoStAR modified cells killed target cells expressing OKT3 equivalently.CoStAR modified TILs do not kill BA/F3 cells expressing FOLR1 alone(FIG. 39B).

Mock or CoStAR modified TIL from 3 ovarian cancer patients wereco-cultured with autologous tumor in the presence of no blocking, MHCI,MHC II or MHC I+MHC II blocking or antibodies or isotype control.Supernatant was assessed for the level of IFNγ release. Normalized tolevels of release without antibody, IFNγ levels are similarly reduced inmock and CoStAR modified TIL, showing that activity is led by endogenousTCR-MHC peptide interactions (FIG. 39C).

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1. A chimeric costimulatory antigen receptor (CoStAR) which comprises: an extracellular binding domain operatively linked to a transmembrane domain, a CD28 signaling domain and a CD40 signaling domain, wherein the at least one extracellular binding domain comprises the amino acid sequence of SEQ ID NO:4, wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 16, and wherein the CD40 signaling domain comprises the amino acid sequence of SEQ ID NO:
 23. 2-16. (canceled)
 17. The CoStAR of claim 1, wherein the extracellular binding domain is operatively linked to the transmembrane domain by a linker and/or a spacer.
 18. The CoStAR of claim 17, wherein the linker comprises from about 5 to about 20 amino acids.
 19. The CoStAR of claim 17, wherein the linker comprises the amino acid sequence AAAGSGGSG (SEQ ID NO:8).
 20. The CoStAR of claim 17, wherein the spacer comprises from about 10 to about 250 amino acids. 21-22. (canceled)
 23. The CoStAR of claim 17, wherein the spacer comprises one or more immunoglobulin domains or an immunoglobulin constant region.
 24. The CoStAR of claim 17, wherein the spacer comprises one or more immunoglobulin domains or an immunoglobulin constant region of SEQ ID NO:13.
 25. The CoStAR of claim 1, wherein the CoStAR further comprises the transmembrane domain of CD28 or CD8.
 26. The CoStAR of claim 1, wherein the CoStAR further comprises the transmembrane domain sequence of SEQ ID NO:11 or SEQ ID NO:12. 27-54. (canceled)
 55. A protein that comprises: a first portion that comprises the amino acid sequence of SEQ ID NO:4 that is linked to; a second portion that comprises a transmembrane domain that is linked to; a third portion that comprises the amino acid sequence of SEQ ID NO: 16 that is linked to; a fourth portion that comprises the amino acid sequence of SEQ ID NO:
 23. 56. The protein of claim 55, wherein the transmembrane domain comprises the transmembrane domain sequence of SEQ ID NO: 11 or SEQ ID NO:
 12. 57. The protein of claim 56, further comprising a linker that comprises the amino acid sequence of SEQ ID NO:
 8. 58. The protein of claim 55, further comprising a signaling domain of SEQ ID NO:
 1. 59. The protein of claim 55, wherein the protein is humanized.
 60. The protein of claim 59, further comprising a VH segment and a VL segment, which are joined together via a linker comprising the sequence of SEQ ID NO:
 62. 61. The protein of claim 60, further comprising a linker, wherein the linker comprises the amino acid sequence of SEQ ID NO:
 62. 62. The CoStAR of claim 1 wherein the CoStAR is humanized.
 63. The CoStAR of claim 62, further comprising a VH segment and a VL segment, which are joined together via a linker comprising the sequence of SEQ ID NO:
 62. 64. The CoStAR of claim 63, further comprising a linker, wherein the linker comprises the amino acid sequence of SEQ ID NO:
 62. 65. A protein that comprises: a first portion that comprises the amino acid sequence of SEQ ID NO:4; a second portion that comprises the amino acid sequence of SEQ ID NO: 11; a third portion that comprises the amino acid sequence of SEQ ID NO: 16; a fourth portion that comprises the amino acid sequence of SEQ ID NO: 23; and a fifth portion that comprises SEQ ID NO: 8, wherein the first portion is linked to the second portion by the fifth portion, wherein the third portion is linked to the second portion, and wherein the fourth portion is linked to the third portion.
 66. A cell comprising: a nucleic acid sequence that encodes a fusion protein, the fusion protein comprising: a first portion that comprises the amino acid sequence of SEQ ID NO:4; a second portion that comprises the amino acid sequence of SEQ ID NO: 11; a third portion that comprises the amino acid sequence of SEQ ID NO: 16; a fourth portion that comprises the amino acid sequence of SEQ ID NO: 23; and a fifth portion that comprises SEQ ID NO: 8, wherein the first portion is linked to the second portion by the fifth portion, wherein the third portion is linked to the second portion, and wherein the fourth portion is linked to the third portion.
 67. The cell of claim 66, wherein the cell is configured to express the fusion protein on a surface of the cell.
 68. The cell of claim 67, wherein the cell comprises an alpha-beta T cell, gamma-delta T cell, T regulatory cell, TIL, NKT cell or NK cell.
 69. The cell of claim 68, wherein the cell comprises a TIL.
 70. The cell of claim 69, wherein the cell co-expresses a CAR or a TCR. 